Pegylated cystathionine beta synthase for enzyme therapy to treat homocystinuria

Abstract

To provide a drug substance for enzyme therapies to treat homocystinuria with higher efficacy and fewer negative effects on the patients than the present therapies.SOLUTION: Provided herein is a drug substance comprising: (a) an isolated cystathionine β synthase (CBS) protein comprising a specific amino acid sequence; and (b) a PEG molecule covalently bonded to the CBS protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compositions and methods for enzyme therapy to treat homocystinuria using the pharmaceutical agents described herein. [Background technology]

[0002] Cystathionine beta-synthase deficiency homocystinuria (CBSDH), also known as classical homocystinuria (HCU) or HCU type 1, is a rare disease affecting both children and adults. CBSDH is a rare autosomal recessive metabolic condition characterized by excess of the compound homocysteine ​​(Hcy) in urine, tissues, and plasma due to reduced or absent activity of the enzyme cystathionine beta-synthase (CBS) (see Non-Patent Document 1; Non-Patent Document 2, each of which is incorporated herein by reference in its entirety).

[0003] The diagnosis of CBSDH can be confirmed by molecular genetic testing of the CBS gene, as described in Non-Patent Document 2. CBS is an enzyme involved in the metabolism of the sulfur amino acid methionine (Met), present in dietary protein (see Non-Patent Document 3, incorporated herein by reference in its entirety). Therefore, CBSDH can also be diagnosed by measuring a significant increase in total methionine concentration in plasma. The finding of these elevated amino acids can be substantiated by detection of decreased CBS enzyme activity or detection of biallelic pathogenic variants in the gene encoding cystathionine beta-synthase (see Non-Patent Document 4, incorporated herein by reference in its entirety). CBS deficiency also leads to decreased levels of cystathionine (Cth) and cysteine ​​(Cys) (see Non-Patent Document 5, incorporated herein by reference in its entirety).

[0004] CBSDH can be suspected based on the following (NPL 4): 1) clinical findings including ectopia lentis (missing eye lens) and / or severe myopia, asthenia (tall and thin), skeletal abnormalities, premature osteoporosis, and / or thromboembolic events, and unexplained developmental delay / intellectual disability; 2) newborn screening for hypermethioninemia, or specifically a positive family history of CBS deficiency, can lead to presymptomatic patient identification (see NPL 6, incorporated herein by reference in its entirety); and 3) family history. There is considerable variability in all of these clinical signs and the age of symptom onset among patients. Current screening methods typically fail to detect newborns with less severe CBS deficiency and only detect a small number of patients with more severe CBSDH (see NPL 7; NPL 8; NPL 9, incorporated herein by reference in their entirety). It is the most common genetic disorder of sulfur metabolism, with an estimated prevalence of 1:200,000 to 1:335,000 worldwide. In the sulfur metabolic pathway, the essential amino acid L-methionine is metabolized to Hcy, followed by a two-step sulfur transfer, first to Cth and finally to Cys. CBS, found primarily in the liver and kidney, catalyzes Hcy to Cth. Excess Hcy can be remethylated to methionine by betaine-Hcy 5-methyltransferase, which can also elevate plasma Met levels.

[0005] Although some patients are diagnosed with the disease shortly after birth, the diagnosis of CBSDH is often made later in life, after the onset of classic clinical symptoms (see Non-Patent Document 7; Non-Patent Document 8, both of which are incorporated herein by reference in their entireties). CBSDH is characterized by developmental delay / intellectual disability, ectopia lentis and / or severe myopia, skeletal abnormalities (excessive height and limb length), and thromboembolic events. There is considerable variability in all of these clinical manifestations between patients.

[0006] Normal total homocysteine ​​(tHcy) levels vary with age, sex, and nutritional status, but typically range between 4.5 and 11 μmol / L (see Non-Patent Document 10, incorporated herein by reference in its entirety). Men tend to have slightly higher (1-2 μmol / L) tHcy levels than women, and a rough doubling of the mean is observed as patients progress from childhood to age 80 (see Non-Patent Document 11, incorporated herein by reference in its entirety). In folic acid-supplemented populations, the upper limit (97.5%) of tHcy levels is approximately 12 μmol / L for adults under 65 years of age and 16 μmol / L for adults over 65 years of age. Many CBSDH patients exhibit severe hyperhomocysteinemia with total tHcy levels exceeding 100 μmol / L, while others exhibit mild to several-fold elevations within the normal range (see Non-Patent Document 12, incorporated herein by reference in its entirety). tHcy levels have been observed to be highly correlated with disease severity (see Non-Patent Document 6, incorporated herein by reference in its entirety).

[0007] CBSDH is characterized by ocular (ectopic lentis and / or severe myopia), skeletal (excessive height, long limbs, scoliosis, and pectus excavatum), vascular (thromboembolic disease), and central nervous system (CNS) (developmental delay / intellectual disability) conditions. There is variable expression of these clinical signs among patients, and all systems may be involved or only one. Some patients have severe, childhood-onset multisystem disease, while those presenting with less severe disease may remain asymptomatic into adulthood. In more severely affected patients, life expectancy is significantly reduced if untreated (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0008] Studies have shown that reduced Hcy levels in CBSDH patients correlate with less severe manifestations of clinical symptoms (see Non-Patent Document 6, which is incorporated herein by reference in its entirety). The pathways by which Hcy levels cause damage to these systems have been widely described (e.g., Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15, each of which is incorporated herein by reference in its entirety), leading to studies to similarly investigate the role of Hcy in the general population.

[0009] Currently, few treatments are available to manage CBSDH. Current treatments target sulfur metabolic pathways through one or more combinations of: 1) dietary modification to reduce protein and / or Met intake; 2) partial or complete supplementation with folic acid, vitamin B12, and vitamin B6; and 3) supplementation with the methyl donor betaine to enhance the enzymatic remethylation of accumulated Hcy to Met. While there are no published studies on the quality of life (QoL) of patients with CBSDH, patients and their caregivers have been observed to suffer from the psychosocial effects of following and managing a highly restrictive and socially isolating diet and are extremely anxious about the long-term medical consequences of the disease. Many patients report a desire to be able to ease their diet without compromising their long-term prospects. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kraus et al., Carmel R, Jacobsen DW (eds.). Homocysteine ​​in Health and Disease. Cambridge, UK: Cambridge University Press; 2001:223-243 [Non-patent document 2] Sacharow et al., Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mefford HC, et al. (eds.). GeneReviews™ [Internet]. Seattle, WA: University of Washington, Seattle, 2017. [Non-patent document 3] Maclean et al. J Biol Chem. 2012;287(38):31994-32005 [Non-patent document 4] Picker et al., Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. January 15, 2004 [updated November 13, 2014]. Pagon RA, Adam MP, Ardinger HH, et al., eds. GeneReviews™ [Internet]. Seattle, WA: University of Washington, Seattle; 1993–2016. Available at ncbi.nlm.nih.gov [Non-patent document 5] Veeranki et al. Int J Mol Sci. 2013 Jul 18;14(7):15074-91 [Non-patent document 6] Yap et al. J Inherit Metab Dis 1998;21:738-47 [Non-Patent Document 7] Huemer et al. J Inherit Metab Dis. 2015 Nov;38(6):1007-19 [Non-patent document 8] Yap, Orphanet Encyclopedia [online periodical]. 2005, pp. 1-13 [Non-Patent Document 9] Schiff et al. Neuropediatrics. 2012 Dec;43(6):295-304 [Non-Patent Document 10] Quest Diagnostics Reference Range;questdiagnostics.com [Non-Patent Document 11] Refsum et al. Clin Chem 2004;50:3-32 [Non-Patent Document 12] Morris et al. J Inherit Metab Dis 2017;40:49-74 [Non-Patent Document 13] Ajith et al. Clin Chim Acta 2015;450:316-321 [Non-Patent Document 14] Sato et al. Bone 2005;36:721-726 [Non-Patent Document 15] Behera et al. J Cell Physiol 2016 Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there is a long felt need in the art for methods of treating CBSDH that are more effective and have fewer negative effects on patients than current treatments. [Means for solving the problem]

[0012] Various embodiments of the present disclosure provide a drug substance comprising: (a) an isolated cystathionine beta synthase (CBS) protein comprising SEQ ID NO: 1; and (b) a PEG molecule covalently attached to the CBS protein. In certain embodiments of the drug substance described herein, the PEG molecule is ME-200GS.

[0013] Various embodiments of the present disclosure provide pharmaceutical compositions comprising a drug substance and a pharmaceutically acceptable adjuvant, diluent, or carrier. In certain embodiments, the formulation is lyophilized. Various embodiments of the present disclosure provide lyophilized formulations that, upon reconstitution, comprise a ready-to-use pharmaceutical agent at a concentration of between about 20-30 mg / ml or about 25 mg / ml in phosphate-buffered saline (PBS); about 11.4 mM disodium hydrogen phosphate; about 137 mM sodium chloride; about 2.70 mM potassium chloride; and about 1.98 mM potassium dihydrogen phosphate. The ready-to-use pharmaceutical agent herein is a liquid pharmaceutical formulation comprising a unit dose of a pharmaceutical composition containing a therapeutically effective amount of a pharmaceutical agent, for example, a PEGylated human truncated CBS protein having the amino acid sequence of SEQ ID NO: 1 (e.g., 20NHSPEG-CBS). The ready-to-use pharmaceutical agent can be provided in a vial or similar container for ease of administration to a subject.

[0014] Various embodiments of the present disclosure provide a lyophilized formulation, wherein upon reconstitution, the reconstituted liquid formulation comprises 20-30 mg or approximately 25 mg of drug substance; 1 mL of water; 2 mg of disodium hydrogen phosphate (dihydrate); 8 mg of sodium chloride; 0.2 mg of potassium chloride; and 0.3 mg of potassium dihydrogen phosphate. Alternatively, the lyophilized formulation can be reconstituted to comprise the drug substance, a buffer, and an excipient. In certain embodiments, the buffer is 15 mM potassium phosphate. In certain embodiments, the excipient is 8% (w / v) trehalose.

[0015] Various embodiments of the present disclosure provide methods of treating homocystinuria in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of a pharmaceutical composition described herein. In certain embodiments, the therapeutically effective amount is a dose selected from the range of about 0.25 mg / kg to about 10 mg / kg. For example, the dose is about 0.33 mg / kg, about 0.66 mg / kg, about 1.0 mg / kg, or about 1.5 mg / kg. Alternatively, the dose is about 2 mg / kg, about 7 mg / kg, and about 10 mg / kg. For example, the dose can be about 0.5 mg / kg. Alternatively, the therapeutically effective amount is a dose selected from the range of about 5.0 mg / kg to about 50 mg / kg and about 10.0 mg / kg to about 25 mg / kg. For example, dosages may be about 0.25 mg / kg, about 0.33 mg / kg, about 0.66 mg / kg, about 1.00 mg / kg, about 1.50 mg / kg, about 2.00 mg / kg, about 3.00 mg / kg, about 4.00 mg / kg, about 5.00 mg / kg, about 6.00 mg / kg, about 7.00 mg / kg, about 8.00 mg / kg, about 9.00 mg / kg, about 10.00 mg / kg, about 11.00 mg / kg, about 12.00 mg / kg, about 13.00 mg / kg, about 14.00 mg / kg, about 15.00 mg / kg, about 16.00 mg / kg, about 17.00 mg / kg, about 18.00 mg / kg, about 19.00 mg / kg, about 20.00 mg / kg, about 21.00 mg / kg, about 22.00 mg / kg, about 23.00 mg / kg, about 24.00 mg / kg, about 25.00 mg / kg, about 26.00 mg / kg, about 27.00 mg / kg, about 28.00 mg / kg, about 29.00 mg / kg, about 30.00 mg / kg, about 31.00 mg / kg, about 32.00 mg / kg, about 33.00 mg / kg, about 34.00 mg / kg, about 35.00 mg / kg, about 36.00 mg / kg, about 37.00 mg / kg, about 38.00 mg / kg, about 39.00 mg / kg, about 39.00 mg / kg, about 39.00 mg / kg, about 39.00 mg / kg, about 39.00 mg / kg, 0.0mg / kg, about 11.0mg / kg, about 12.0mg / kg, about 13.0mg / kg, about 14.0mg / kg, about 15.0mg / kg, about 16.0mg / kg, about 17.0mg / kg, about 18.0mg / kg, about 19.0mg / kg, about 20.0mg / kg, about 21.0mg / kg, about 22.0mg / kg, about 23.0mg / kg, about 24 .0mg / kg, about 25.0mg / kg, about 26.0mg / kg, about 27.0mg / kg, about 28.0mg / kg, about 29.0mg / kg, about 30.0mg / kg, about 31.0mg / kg, about 32.0mg / kg, about 33.0mg / kg, about 34.0mg / kg, about 35.0mg / kg, about 36.0mg / kg, about 37.0mg / kg, about 38 0.0 mg / kg, about 39.0 mg / kg, about 40.0 mg / kg, about 41.0 mg / kg, about 42.0 mg / kg, about 43.0 mg / kg, about 44.0 mg / kg, about 45.0 mg / kg, about 46.0 mg / kg, about 47.0 mg / kg, about 48.0 mg / kg, about 49.0 mg / kg, and about 50.0 mg / kg.

[0016] In certain embodiments, the method further comprises administering to the subject at least one selected from the group consisting of pyridoxine, vitamin B6, and betaine. In certain embodiments, the subject is on a methionine (Met) restricted diet. In certain embodiments, the method further comprises administering an antiplatelet agent. In certain embodiments, the antiplatelet agent is a warfarin blood thinner or anticoagulant. In certain embodiments, the administering step occurs about once every three days. In certain embodiments, the administering step occurs about once a day. In certain embodiments, the administering step occurs about once a week. In certain embodiments, the administering step is repeated for about six weeks. In certain embodiments, the administering step is repeated for about three months. In certain embodiments, the administering step is repeated for about six months. In certain embodiments, the administering step is repeated for more than six months. In certain embodiments, the administering step is repeated for the remainder of the subject's life.

[0017] Various embodiments of the present disclosure provide methods of lowering homocysteine ​​(Hcy) levels in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of a pharmaceutical composition described herein.

[0018] In certain embodiments, Hcy levels are less than about 80 μM after the administering step. In certain embodiments, Hcy levels are reduced by up to 10% after the administering step. In certain embodiments, Hcy levels are reduced by up to 20% after the administering step. In certain embodiments, Hcy levels are reduced by up to 30% after the administering step. In certain embodiments, Hcy levels are reduced by up to 40% after the administering step. In certain embodiments, Hcy levels are reduced by up to 50% after the administering step. In certain embodiments, Hcy levels are reduced by up to 60% after the administering step. In certain embodiments, Hcy levels are reduced by up to 70% after the administering step. In certain embodiments, Hcy levels are reduced by up to 80% after the administering step. In certain embodiments, Hcy levels are reduced by up to 90% after the administering step. In certain embodiments, Hcy levels are in the range of about 10 μM to about 20 μM after the administering step. In certain embodiments, Hcy levels are less than 10 μM after the administering step, hi certain embodiments, Hcy levels are about 55 μM after the administering step.

[0019] In certain embodiments, the therapeutically effective amount is a dose selected from the range of about 0.25 mg / kg to about 10 mg / kg. For example, the dose can be about 0.33 mg / kg, about 0.66 mg / kg, about 1.0 mg / kg, about 1.50 mg / kg, about 2.0 mg / kg, about 7.0 mg / kg, or about 10 mg / kg. In certain embodiments, the dose is less than 10 mg / kg.

[0020] In certain embodiments, the method further comprises administering to the subject at least one selected from the group consisting of pyridoxine, vitamin B6, and betaine. In certain embodiments, the subject is on a methionine (Met) restricted diet. In certain embodiments, the method further comprises administering an antiplatelet agent. In certain embodiments, the antiplatelet agent is a warfarin blood thinner or anticoagulant. In certain embodiments, the administering step occurs about once every three days. In certain embodiments, the administering step occurs about once a day. In certain embodiments, the administering step occurs about once a week. In certain embodiments, the administering step is repeated for about six weeks. In certain embodiments, the administering step is repeated for about three months. In certain embodiments, the administering step is repeated for about six months. In certain embodiments, the administering step is repeated for more than six months. In some embodiments, the administering step is repeated for the remainder of the subject's life.

[0021] Various embodiments of the present disclosure provide methods of increasing cysteine ​​(Cys) levels in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of a pharmaceutical composition described herein.

[0022] Various embodiments of the present disclosure provide a method for increasing cystathionine (Cth) levels in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of the pharmaceutical composition described herein.

[0023] Various embodiments of the present disclosure provide methods for treating, alleviating, or preventing negative clinical outcomes associated with the ocular, skeletal, vascular, and / or central nervous systems of a subject, comprising administering to the subject a therapeutically effective amount of a formulation of a pharmaceutical composition described herein.

[0024] The above and other objects, features and advantages will become apparent from the following description of specific embodiments of the present disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale; instead, emphasis is placed upon illustrating the principles of various embodiments of the present disclosure. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is a flow diagram of the PEGylation process utilized in certain embodiments described herein. [Figure 2] Diagram of entry conditions in the puzzle box test for each trial during the 3-day test period. [Figure 3] Diagram showing preclinical study results in a mouse model of homocystinuria employing long-term continuous treatment with 20NHS PEG-CBS (referred to in the figure as "PEG CBS C15S") on a background of varying diets (standard methionine diet ("STD"), high methionine diet ("HMD"), and / or low or restricted methionine diet ("MRD")). DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Introduction Cystathionine beta-synthase deficiency homocystinuria (CBSDH) is characterized by elevated levels of plasma homocysteine ​​(Hcy) along with high levels of Met and decreased concentrations of cysteine ​​(Cys). (See Yap S., Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [Regular Online] 2005; Non-Patent Document 12; NORD, Kraus JP. Homocystinuria due to cystathionine beta-synthase deficiency. NORD [Regular Online] 2017, each of which is incorporated herein by reference in its entirety.) To date, over 180 distinct mutations in the CBS gene associated with CBSDH have been identified (see Human Genome Mutation Database. 2017. Ref Type: available at online source.hgmd.cf.ac.uk / ac / index.php, incorporated herein by reference in its entirety). Homocysteine ​​(Hcy) is a naturally occurring amino acid that, together with serine, serves as a substrate for the CBS enzyme. CBS governs the unidirectional flow of sulfur from methionine (Met) to cysteine ​​(Cys) by operating at the crossroads of the transmethylation, transsulfuration, and remethylation pathways (see Non-Patent Document 3, incorporated herein by reference in its entirety). Native CBS is activated by the binding of the allosteric activator S-adenosylmethionine (SAM) and catalyzes the β-substitution reaction in which serine condenses with Hcy to form cystathionine (Cth) in a pyridoxal-5'-phosphate (pyridoxine or vitamin B6)-dependent manner. Cystathionine γ-lyase (CGL), which operates downstream of CBS, uses Cth as a substrate to generate Cys. Thus, proper function of CBS is important for regulating the metabolism of Hcy, Met, and Cys.

[0027] The severity and presentation of CBSDH signs and symptoms vary widely among patients (see Karaca et al., Gene 2014;534:197-203; Trondle et al., Acta Med Austriaca 2001;28:145-151; Kluijtmans et al., Am J Hum Genet 1999;65:59-67, each of which is incorporated herein by reference in its entirety). Many patients present with severe hyperhomocysteinemia, with total homocysteine ​​(tHcy) levels exceeding 100 μmol / L, while others present with tHcy elevations ranging from mild to several-fold normal (see Non-Patent Document 12, incorporated herein by reference in its entirety). Significantly elevated tHcy levels typically correlate with severe presentation, while lower levels typically correlate with milder forms of the disease. Normal tHcy levels vary with age and nutritional status, but typically range between 10 and 15 μmol / L.

[0028] CBSDH is generally classified according to whether affected individuals respond to total homocysteine ​​(tHcy)-lowering treatment with pyridoxine (vitamin B6), a CBS enzyme cofactor required for normal CBS function (see Mudd et al., Am J Hum Genet 1985;37:1-31; Abbott et al., Am J Med Genet 1987;26:959-969, incorporated herein by reference in their entireties). Patients who respond to pyridoxine typically have lower tHcy levels, resulting in milder forms of the disorder. These patients may present with only one or a few CBSDH symptoms later in life, and many remain undiagnosed. As a result, highly pyridoxine-responsive patients are considered underrepresented in most studies.

[0029] Retrospective studies have shown that patients (treated or untreated) with the highest tHcy levels present with more severe symptoms and earlier in life (see Non-Patent Document 6; Mudd et al. Am J Hum Genet 1985;37:1-31, both of which are incorporated herein by reference in their entireties). Untreated individuals with elevated tHcy levels typically exhibit growth retardation, thromboembolism, severe myopia with subsequent dislocation of the eye lens, osteoporotic fractures, Marfanoid diathesis (especially elongation of long bones), and psychiatric abnormalities, including learning difficulties. (See Yap S., Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [Regular Online] 2005; Non-Patent Document 12; NORD, Kraus J. P., Homocystinuria due to cystathionine beta-synthase deficiency. NORD [Regular Online] 2017, each of which is incorporated herein by reference in its entirety.) Some patients with elevated tHcy levels have a severe, childhood-onset multisystem disorder. In more severely affected patients, life expectancy is significantly reduced if untreated (see, e.g., 1999, J. Med. 1999, 2001, pp. 111-113, which is incorporated herein by reference in its entirety).

[0030] Normal human plasma contains less than 16 mM Hcy-derived compounds, measured as tHcy and consisting of free thiol homocysteine ​​(Hcy-SH or fHcy), disulfides (such as homocysteine-cysteine ​​and homocysteine), and protein-bound homocysteine ​​(see Ueland; Nord Med 1989;104:293-298; Mudd et al. N Engl J Med 1995;333:325; Mudd et al. Arterioscler Thromb Vasc Biol 2000;20:1704-1706, each of which is incorporated herein by reference in its entirety). Many of the pathophysiological effects depend on the presence of sulfhydryl groups in Hcy (Yap S., Homocystinuria due to cystathionine beta-synthase deficiency, Orphanet Encyclopaedia [regular online] 2005; Ueland et al.; Nord, each of which is incorporated herein by reference in its entirety). Med 1989;104:293-298; Mudd et al. N Engl J Med 1995;333:325), and the distinction between the sulfhydryl form (homocysteine; Hcy) and the disulfide form (homocysteine) (see Yap S., Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [regular online] 2005, incorporated herein by reference in its entirety).

[0031] CBS is primarily expressed in the liver, pancreas, kidney, and brain (see Non-Patent Document 12, which is incorporated herein by reference in its entirety). The catalytic domain binds to pyridoxal 5'-phosphate (a cofactor also known as pyridoxine or vitamin B6), and the regulatory domain binds to SAM (an allosteric activator).

[0032] Insufficient levels of CBS enzyme activity block the transsulfuration pathway at the first step, resulting in Hcy accumulation, elevated SAH and Met levels, and decreased Cth and Cys levels. These clinical evidences, along with the dysregulated Met metabolites outlined herein, demonstrate that elevated Hcy (often measured clinically as plasma tHcy) is most strongly implicated in the pathophysiology of CBSDH.

[0033] Higher-than-normal Hcy levels modify sulfhydryl groups on proteins, preventing proper protein cross-linking and leading to structural abnormalities across multiple body systems. Elevated Hcy levels also impair intracellular signaling, leading to endothelial dysfunction and ultimately thromboembolism and vascular disease. In CBSDH, Hcy accumulation leads to ocular, skeletal, vascular, and psychological manifestations.

[0034] The diagnosis of CBSDH is sometimes confirmed by molecular genetic testing of the CBS gene (see Non-Patent Document 2, incorporated herein by reference in its entirety). Current screening methods typically fail to detect newborns with less severe CBS defects and detect only a small number of patients with more severe CBSDH (see Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9).

[0035] The commonly preferred measurement for determining Hcy levels in clinical samples is tHcy, which includes free Hcy as well as protein-bound or disulfide-bound Hcy. Normal tHcy levels vary with age, sex, and nutritional status, but typically range between 4.5 and 11 μM (QUEST DIAGNOSTICS™ reference range). Many CBSDH patients exhibit severe hyperhomocystinuria with total tHcy levels exceeding 100 μM, while others exhibit mild to several-fold elevations within the normal range (see Non-Patent Document 12, incorporated herein by reference in its entirety). tHcy levels are highly correlated with disease severity (see Non-Patent Document 6).

[0036] Studies have shown that reduced Hcy levels in CBSDH patients correlate with less severe manifestations of clinical symptoms (see, e.g., J. Med. 2001, 11:111-112; Yap et al., Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):2080-5, both of which are incorporated herein by reference in their entireties). The pathways by which homocysteine ​​levels cause damage to these systems have been extensively described (see, e.g., J. Med. 2001, 11:111-112; Saha et al., ... et al.) FASEB J 2016;30:441-456), leading to studies investigating the role of Hcy in the general population, uncovering a significant pathological role for Hcy in disease.

[0037] One goal of treatment with the pharmaceutical agents described herein is to increase CBS enzyme activity in the circulation, resulting in improved metabolic control, thereby ameliorating the clinical manifestations of the disease and slowing or preventing further deterioration. High molecular weight compounds such as enzymes have limited tissue penetration capabilities and are therefore primarily present in the plasma. These proteins typically remain in the circulation for short periods of time as they are removed from the bloodstream by several mechanisms (Vugmeyster et al., 2004, incorporated herein by reference in its entirety). (See Kang et al., World J Biol Chem. 2012;3(4):73-92). Ideally, administered CBS would maintain high activity in plasma for a sufficient time to have a steady effect on sulfur amino acid metabolism. This goal can be achieved by PEGylation, the addition of PEG moieties to the protein surface. Protein PEGylation is a widely accepted strategy that has been shown to increase protein stability and size, reduce renal excretion, while minimizing proteolysis, immune response, and antigenicity (See Kang et al., 2009;14(2):363-380, incorporated herein by reference in its entirety). The pharmaceutical preparations described herein are PEGylated htCBS C15S enzymes formulated for administration to a subject and designed for long-term systemic exposure.

[0038] A. Clinical Manifestations of Homocystinuria There is significant evidence pointing to a causal effect of elevated tHcy levels and negative clinical outcomes in the four systems commonly affected in patients with CBSDH (ophthalmic, skeletal, cardiovascular, and neurological). These data are further complemented by studies in the general population demonstrating a strong relationship between mildly elevated levels of tHcy and negative outcomes.

[0039] 1.Eye Ocular abnormalities can be an early clinical sign of CBSDH. Many individuals develop displacement of the eye's lens from the center of the eyeball (ectopia lentis). Affected individuals also usually develop severe myopia (nearsightedness) and iridokyphosis (trembling of the colored portion of the eye). Ectopia lentis and myopia usually develop after the age of one year, and in untreated individuals before the age of ten (see Mudd et al. Am J Hum Genet 1985;37:1-31, incorporated herein in its entirety). Other less frequently occurring ocular abnormalities include cataracts, optic nerve degeneration, and glaucoma. Some individuals may have retinal detachment, which can cause blurred vision or the appearance of "floaters" in the field of vision (see Burke et al., Am J Hum Genet 1985;37:1-31, incorporated herein in its entirety). al.) Br J Ophthalmol, 1989;73(6):427-31).

[0040] Elevated Hcy levels are a strong independent risk factor for ocular complications, particularly lens dislocation, in CBSDH patients and the general population (see Mudd et al., Am J Hum Genet 1985;37:1-31; Non-Patent Document 13; Mulvihill et al., J AAPOS 2001;5:311-315, which are incorporated herein by reference in their entirety). Even with prescribed pharmacological and dietary interventions, the majority of CBSDH patients ultimately develop ocular complications. Lowering Hcy levels has been observed to delay and potentially prevent lens dislocation in CBSDH patients (see Non-Patent Document 6, which is incorporated herein by reference in its entirety).

[0041] 2. Central nervous system Developmental delays, such as mental retardation, and learning problems may also be early signs of CBSDH, occurring between the ages of 1 and 3 years (Screening, Technology, 2004, pp. 111-113, incorporated herein by reference in its entirety). and Research in Genetics (STAR-G) Project. 2016. Available at: Homocystinuria.newbornscreening.info; National Institutes of Health National Institutes of Health (NIH), US National Library of Medicine, Genetics Home Reference.Homocystinuria.2016.ghr.nlm.nih.gov). Intelligence quotients (IQs) in individuals with CBSDH have been reported to range from 10 to 138. Patients with the highest tHcy levels are more likely to have a lower IQ (mean IQ of 57 if untreated) compared with less severely affected patients (mean IQ of 79) (see Non-Patent Document 2, incorporated herein by reference in its entirety).

[0042] Seizures occur in approximately 20% of untreated individuals with CBSDH (see Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety). Many individuals have psychiatric problems, including personality disorders, anxiety, depression, obsessive-compulsive behavior, and psychotic episodes (see Non-Patent Document 2). Extrapyramidal symptoms, such as dystonia, may also occur (see Screening, Technology and Research in Genetics (STAR-G) Project. 2016. Available at: Homocystinuria.newbornscreening.info, incorporated herein by reference in its entirety).

[0043] Studies have shown that early reduction of Hcy levels induced by a low-Met diet, folic acid / B vitamin supplementation, and / or pyridoxine and betaine therapy can slow, and sometimes prevent or reverse, the progression of various neurological disorders and allow for normal IQ development in CBSDH patients (see El Bashir et al. JIMD Rep 2015;21:89-95; Yap et al. J Inherit Metab Dis 2001;24:437-447, incorporated herein by reference in their entireties). The association between elevated Hcy levels and central nervous system (CNS) symptoms, including mental retardation, neurodegenerative diseases, seizures, dystonia, psychosis, cognitive impairment, dementia, and depression, has been well documented in CBSDH patients and the general population (Abbott et al. Am J Med Genet 1987;26:959-969; Schimke et al., 2004, incorporated herein by reference in their entirety). al.) JAMA 1965;193:711-719; Herrmann et al. Clin Chem Lab Med 2011;49:435-441).

[0044] 3. Skeletal system Individuals with CBSDH frequently develop a variety of skeletal abnormalities. Affected individuals are often tall and thin, with a "Marfan-like" physique, including thinning and elongation of the long bones (arachnopodiatry), knees that bow inward so that they touch when the legs are straightened ("genu valgum" or knock-knees), highly arched feet (pes cavus), an abnormal sideways curvature of the spine (scoliosis), and an abnormally protruding chest (pectus carinatum) or an abnormally sunken chest (pectus excavatum). By their teenage years, 50% of individuals show signs of osteoporosis (see Screening, Technology and Research in Genetics (STAR-G) Project. 2016. Available at Homocystinuria.newbornscreening.info, incorporated herein by reference in its entirety). CBSDH is associated with an increased risk of osteoporotic fractures, which may be due in part to low bone mineral density (see Mudd et al. Am J Hum Genet 1985;37:1-31; Weber et al. Mol Genet Metab 2016;117:351-354, incorporated herein by reference in their entireties).

[0045] A study of 25 Irish CBSDH patients followed over a 25-year period found that the risk of skeletal abnormalities was significantly lower in patients who were highly compliant with Hcy-lowering treatment compared with non-compliant patients (see Non-Patent Document 6, incorporated herein by reference in its entirety).

[0046] 4.Cardiovascular system The relationship between CBSDH and vascular disease was first demonstrated in 1985 in an epidemiological study in patients with moderately to severely elevated Hcy levels due to homozygous CBSDH (Mudd et al., Am J Hum, 2005, 144(1):145-150, which is incorporated herein by reference in its entirety). Genet 1985;37:1-31). Thromboembolism is the most serious, often life-threatening, complication of CBSDH and can affect any blood vessel. Thromboembolism is a major cause of morbidity and premature mortality in patients with CBSDH (see Yap et al., Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):2080-5, incorporated herein by reference in its entirety).

[0047] The risk of thromboembolic events was approximately 25% by age 16 and 50% by age 29. Several reports have described how treatments to lower tHcy levels significantly reduced the incidence of vascular events, a major cause of morbidity, in patients with CBSDH (see Wilcken et al. J Inherit Metab Dis 1997;20:295-300; Yap et al. Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):2080-5, which are incorporated herein by reference in their entireties). Since then, several other studies have demonstrated an increased risk of vascular events, particularly venous thrombosis, in patients with CBSDH (see Kelly et al. Neurology 2003;60:275-279; Magner et al. J Neurology 2003;60:275-279, which are incorporated herein by reference in their entireties). See Inherit Metab Dis 2011;34:33-37).

[0048] 5. Additional Manifestations Although less common, several additional findings have been reported in patients with CBSDH, including extremely fine, brittle skin, easily damaged hair, skin discoloration (hypopigmentation), and a rash on the cheeks (malar flushing). Some individuals may develop fatty changes in the liver, protrusion of a portion of the intestine through a laceration in the abdominal wall (inguinal hernia), or inflammation of the pancreas. An abnormal front-to-back curvature of the spine (kyphosis) and collapsed lungs (spontaneous pneumothorax) have also been reported in individuals with CBSDH (see Non-Patent Document 8, incorporated herein by reference in its entirety).

[0049] In summary, MRD alone is effective in correcting multiple symptoms of HCU, despite failing to lower plasma Hcy concentrations below recommended levels, leading to increased anxiety and decreased bone mineralization. On the other hand, enzyme therapy with 20NHS PEG-CBS described herein reduced plasma Hcy concentrations below the suggested threshold of 100 μM and corrected all monitored symptoms of HCU. Furthermore, 20NHS PEG-CBS maintained its efficacy under Met restriction, resulting in a fully normalized plasma biochemistry profile. By extrapolating these data to human patients, our results establish that 20NHS PEG-CBS as a single, lifelong therapy can be effective in preventing and correcting the clinical symptoms of HCU. Additionally, treatment with 20NHS PEG-CBS allows for the elimination of Met / dietary restrictions, which, in turn, should substantially improve the quality of life of HCU patients and their families.

[0050] II. Composition A. Native human CBS enzyme The CBS intact native enzyme is a tetramer containing four identical monomers, each 63 kDa in size, organized into three functional domains. The first is an N-terminal region of approximately 70 amino acids that binds heme and is thought to function in redox sensing and / or enzyme folding. The second is a central domain that contains the catalytic core and exhibits a type II family PLP (pyridoxal-5'-phosphate)-dependent enzyme fold. The coenzyme PLP is buried deep in a cleft between the N- and C-terminal domains. The third region is a C-terminal regulatory domain consisting of a tandem pair of CBS motifs that activates the enzyme upon binding S-adenosylmethionine (SAM). Removal of the regulatory region generates a constitutively active enzyme (Miles et al., 2004, incorporated herein by reference in its entirety). et al.) J Biol Chem. July 16, 2004;279(29):29871-4).

[0051] The pyridoxal-5'-phosphate (PLP)-dependent enzyme fold contains a heme group. This enzyme catalyzes the PLP-dependent beta-substitution reaction that condenses L-homocysteine ​​with L-serine to form L-cystathionine. This enzyme is allosterically regulated by S-adenosyl-L-methionine (Ado-Met) binding to the C-terminal regulatory domain, resulting in conformational rearrangement of these domains and the release of an autoinhibitory block. CBS activation can also be achieved by completely removing the C-terminal regulatory domain to generate a constitutively active dimeric form of the enzyme (see Miles et al. J Biol Chem. 2004 Jul 16;279(29):29871-4; Ereno-Orbea et al. Proc Natl Acad Sci USA 111(37), E3845-3852 (2014), each of which is incorporated herein by reference in its entirety).

[0052] The active substance in the pharmaceutical products described herein is a recombinant human truncated CBS protein (htCBS C15S) having a cysteine ​​to serine substitution at amino acid position 15 of the protein compared to the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing and SEQ ID NO: 2 in WO 2017 / 083327 (incorporated herein in its entirety), which represents a native CBS protein modified by the addition of polyethylene glycol (PEG). This enzyme is also known as htCBS C15S. In certain embodiments, the drug substance htCBS C15S has the amino acid sequence of SEQ ID NO: 1.

[0053] This form of the enzyme has a high tendency toward aggregation, imposing significant limitations on the production and manufacture of human CBS (hCBS). PEGylated htCBS C15S (including "20NHS PEG-CBS" as defined herein) is engineered to form dimers, rather than tetramers, which are less susceptible to aggregation. High-molecular-weight compounds, such as enzymes, are removed from the circulation by proteolytic degradation and various clearance mechanisms (see Vugmeyster et al., World J Biol Chem. 2012;3(4):73-92, incorporated herein in its entirety). PEGylation is known to increase protein stability, reduce renal excretion, and minimize proteolysis and immunogenicity (see Kang et al., 2009;14(2):363-380, incorporated herein in its entirety). These structural modifications make the pharmaceutical preparations described herein comprising PEGylated htCBS C15S a better candidate than native hCBS for enzyme therapy (ET) for CBSDH.

[0054] Native CBS is an intracellular enzyme, and there is no known mechanism for importing the enzyme from the extracellular environment to its primary intracellular site of action. However, PEGylated htCBS C15S acts extracellularly. Unlike native endogenous CBS, PEGylated htCBS C15S corrects metabolic disorders by acting directly in the circulation and indirectly in tissues, and does so without requiring SAM for activation. The native htCBS enzyme is activated intracellularly upon binding of S-adenosylmethionine (SAM) to its C-terminal regulatory domain. However, in both patients and healthy individuals, circulating SAM levels are far lower than those required for CBS activation (see Stabler et al., Metabolism, 2002, 51(8):981-8, incorporated herein by reference in its entirety). Therefore, administration of native CBS to the circulation would be ineffective, as CBS would not be activated. PEGylated htCBS C15S remains in the circulation and does not enter cells, but has been engineered to bypass the requirement for SAM activation by removal of the CBS C-terminal regulatory domain, rendering the enzyme constitutively active.

[0055] B. Enzyme therapy (ET) PEGylated htCBS C15S is a PEGylated truncated hCBS with a cysteine ​​to serine substitution at position 15 for ET to treat CBSDH. This modification optimizes the enzyme to form dimers rather than tetramers and is constitutively active.

[0056] PEGylated htCBS C15S replaces the defective CBS activity, thereby lowering plasma, urinary, and tissue levels of homocysteine ​​(Hcy) and methionine (Met), increasing cystathionine (Cth), and normalizing cysteine ​​(Cys) levels in patients with CBSDH. Reduction of total Hcy (tHcy) levels is a current treatment target (see Non-Patent Document 12, incorporated herein by reference in its entirety) and is strongly correlated with improved clinical (ophthalmic, skeletal, vascular, and neurological) outcomes (Non-Patent Document 8, incorporated herein by reference in its entirety).

[0057] PEGylated htCBS C15S is a recombinant form of the native human CBS enzyme and is produced in Escherichia coli (E. coli) bacteria. The DNA sequence of native human CBS (SEQ ID NO: 3 in the Sequence Listing and SEQ ID NO: 1 in WO 2017 / 083327, the entire contents of which are incorporated herein by reference) was genetically modified to remove the C-terminal regulatory region (amino acids 414-551) (SEQ ID NO: 4 in the Sequence Listing and SEQ ID NO: 3 in WO 2017 / 083327) to form human truncated CBS. The DNA sequence of human truncated CBS was further modified to introduce a T-to-A point mutation at position 43 of the DNA coding region, resulting in a cysteine-to-serine substitution at position 15 of the translated protein, generating human truncated CBS C15S (htCBS C15S) (SEQ ID NO: 5 in the Sequence Listing and SEQ ID NO: 13 in WO 2017 / 083327). This change reduces aggregation and allows for batch-to-batch consistency compared to native hCBS.

[0058] This enzyme is further engineered during expression in E. coli bacteria to remove the first Met from the protein set forth in SEQ ID NO: 1. After its purification, the htCBS C15S enzyme is further modified by PEGylation with N-hydroxysuccinimide ester-functionalized 20 kDa PEG moieties, which react with primary amines on the surface of the protein. An approximate average of 5.1 PEG molecules are attached to each monomer unit of the enzyme, yielding a heterodimeric product with an average molecular weight of 290 kDa.

[0059] C. PEGylation of htCBS C15S to generate 20NHS PEG-CBS ME-200GS (also referred to as methoxy-PEG-CO(CH2)3COO-NHS) is used herein to PEGylate htCBS C15S:

[0060] [ka]

[0061] ME-200GS has a molecular weight of 20 kDa and the chemical name α-succinimidyloxyglutaryl-ω-methoxy, polyoxyethylene. ME-200GS targets free amines on the surface of htCBS C15S. An amide bond is formed between PEG and a lysine residue on htCBS C15S. The resulting molecule is referred to throughout this disclosure as "20NHS PEG-CBS," a PEGylated human cystathionine beta synthase molecule with a truncated C15S mutation, provided in SEQ ID NO: 1.

[0062] Tangential flow filtration (TFF) with a 100 kDa molecular weight cutoff and 15 volume exchanges is used to deplete free PEG and other PEGylated impurities prior to formulation into PBS buffer. Ammonium ions and all other process-related substances, including imidazole, Triton X-100, and the NHS group released from PEG during PEGylation, are expected to be at trace levels after two cycles of the large cutoff TFF diafiltration step.

[0063] PEG ME-200GS is manufactured by NOF Corporation under cGMP conditions according to the process flow diagram provided in Figure 1. All raw materials used in the manufacture of the ME-200GS product are synthetic or inorganic in nature. Impurities that may potentially be present in the PEG ME-200GS raw materials are small molecules.

[0064] D. Post-translational Modification Post-translational modifications require additional bioprocessing steps to separate modified and unmodified polypeptides, which can increase the cost and reduce the efficiency of biologic production. Thus, in some embodiments, the production of a polypeptide agent in a cell is enhanced by modulating the expression of a target gene encoding a protein that affects the post-translational modification. In additional embodiments, the production of a biologic is enhanced by modulating the expression of a first target gene encoding a protein that affects a first post-translational modification and modulating the expression of a second target gene encoding a protein that affects a second post-translational modification.

[0065] Furthermore, proteins expressed in prokaryotic or eukaryotic cells may undergo several post-translational modifications that may impair the production and / or structure, biological activity, stability, homogeneity, and / or other properties of the biological product. Many of these modifications occur spontaneously during cell growth and polypeptide expression and can occur at several sites, including the peptide backbone, amino acid side chains, and amino and / or carboxyl termini of a given polypeptide. In addition, a given polypeptide may contain several different types of modifications. For example, proteins expressed in bacterial cells such as E. coli may be subject to acetylation, histone clipping, carboxylation, and / or deamidation (see Yang et al., PNAS 111(52)E5633-E5642 (2014), incorporated herein by reference in its entirety). For example, proteins expressed in avian and mammalian cells, such as Chinese hamster ovary (CHO) cells, can be subject to acetylation, carboxylation, gamma-carboxylation, histone clipping, deamidation, N-terminal glutamine cyclization and deamidation, and asparagine deamidation.

[0066] In some embodiments, protein production is enhanced by modulating the expression of a target gene encoding a protein involved in protein deamidation. Proteins can be deamidated via several pathways, including cyclization and deamidation of N-terminal glutamine and deamidation of asparagine. Thus, in one embodiment, the protein involved in protein deamidation is N-terminal asparagine aminohydrolase. Protein deamidation can lead to altered structural characteristics, reduced potency, reduced biological activity, reduced efficacy, increased immunogenicity, and / or other undesirable properties, which can be measured by several methods, including, but not limited to, charge-based protein separation using ion exchange chromatography, HPLC, isoelectric focusing, capillary electrophoresis, native gel electrophoresis, reversed-phase chromatography, hydrophobic interaction chromatography, affinity chromatography, mass spectrometry, or L-isoaspartyl methyltransferase.

[0067] In some embodiments, the protein that affects protein secretion is a molecular chaperone selected from the group consisting of Hsp40, HSP47 (serpin peptidase inhibitor, clade H; also known as heat shock protein 47), HSP60, Hsp70, HSP90, HSP100, protein disulfide isomerase, peptidyl prolyl isomerase, calnexin, Erp57 (protein disulfide isomerase family A, member 3), and BAG1. In some embodiments, the protein that affects protein secretion is selected from the group consisting of γ-secretase, p115, signal recognition particle (SRP) protein, secretin, and a kinase (e.g., MEK).

[0068] It is contemplated that further optimization can be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing the created sequences by moving from this point through longer or shorter size windows above or below target RNA.This approach to creating new candidate targets can be coupled with testing the effectiveness of RNA effector molecules based on target sequences in inhibition assays known in the art or described herein, which can lead to further improvements in inhibition efficiency.Furthermore, such optimized sequences can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or discussed herein to further optimize molecules as expression inhibitors (for example, increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0069] E. Stability The drug substance or drug product is stable at various temperatures and storage conditions. In some embodiments, the drug substance or drug product is stable when stored at -65°C and -20°C. Alternatively, the drug substance or drug product may be stable when stored at a temperature ranging from about 2°C to about 8°C. Alternatively, the drug substance or drug product may be stable when stored at a temperature ranging from 25°C ± 2°C. For example, the drug substance or drug product remains stable between 20°C and 25°C. In certain embodiments, the drug substance or drug product is stable under reducing conditions. In certain embodiments, the drug substance or drug product is stable under non-reducing conditions. In some embodiments, the drug substance or drug product remains stable for at least 2 days, at least 7 days, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or at least 12 months. For example, the drug substance or drug product remains stable during storage for about 2 days. For example, the drug substance or drug product remains stable during storage for about 7 days. For example, the drug substance or drug product remains stable during storage for about 1 month. For example, the drug substance or drug product remains stable during storage for about 2 months. For example, the drug substance or drug product remains stable during storage for about 3 months. For example, the drug substance or drug product remains stable during storage for about 6 months. For example, the drug substance or drug product remains stable during storage for about 12 months. For example, the drug substance or drug product remains stable during storage for about 18 months.

[0070] In some embodiments, the drug substance or drug product remains stable for up to 18 months of storage at −65° C. In some embodiments, the drug substance or drug product remains stable for up to 3 months of storage between about 2° C. and about 8° C. In some embodiments, the drug substance or drug product remains stable for up to 1 month of storage at 25° C.±2° C.

[0071] In some embodiments, the drug substance or drug product remains stable for at least three freeze-thaw cycles.In some embodiments, the drug substance or drug product remains stable for up to six freeze-thaw cycles.For example, the drug substance or drug product remains stable for five freeze-thaw cycles.In certain embodiments, the drug product is stable after being ejected from a syringe.

[0072] III. Pharmaceutical Compositions The pharmaceutical preparations described herein, comprising PEGylated htCBS C15S, are intended to lower homocysteine ​​levels and normalize cysteine ​​levels in CBSDH patients, thereby restoring metabolic control and improving the clinical manifestations of the disease. C15S is produced by recombinant technology using Escherichia coli (E. coli) BL21(DE3) and formulated as a sterile drug product in phosphate-buffered saline. The drug product is intended for administration by subcutaneous (SC) injection.

[0073] Circulating PEGylated htCBS C15S activity also improved or even completely normalized metabolite profiles in tissues (see WO 2017 / 083327, incorporated herein by reference in its entirety). Thus, pharmaceuticals do not necessarily need to be delivered to their natural intracellular environment.

[0074] The pharmaceuticals reduce the accumulation of toxic Hcy in the circulation, urine, and tissues of CBSDH patients; normalize Cys levels in the circulation and tissues; increase Cth levels in the circulation and tissues; and / or prevent, delay, and / or reverse the onset of CBSDH manifestations. The pharmaceuticals achieve at least one of these benefits while allowing patients to enjoy a normal diet. Indeed, increased Cth activity even with a normal diet (e.g., 4.0 g / kg MET) has been observed to be evidence of increased activity and / or decreased renal excretion of the pharmaceuticals.

[0075] The 20NHS PEG-CBS drug substance was formulated at a concentration between 20-30 mg / mL or approximately 25 mg / mL in phosphate-buffered saline (PBS) containing disodium hydrogen phosphate (dihydrate) (11.4 mM), sodium chloride (137 mM), potassium chloride (2.7 mM), and potassium dihydrogen phosphate (1.98 mM) prepared with water for injection (WFI). The drug substance was freely soluble in aqueous solution.

[0076] The molecular weights of the drug substance calculated from the isotopically averaged molecular weights from SEC / UV / MS are 45.290 kDa for the monomer and 90.58 kDa for the dimer. All batches were clear, dark red liquids with virtually no visible particles. Furthermore, SDS-PAGE and Western blot analysis performed under both reducing and non-reducing conditions provided consistent results for each batch. Using each of these methods, unique, uniform, and consistent patterns of PEGylated variants were demonstrated. Concomitant medications, including anticoagulants, vitamin and mineral supplements, betaine, and antidepressants, can also be combined with the pharmaceutical compositions described herein to enhance the efficacy of the pharmaceutical compositions.

[0077] IV. Formulations For the above-mentioned therapeutic uses, the administered dosage will vary depending on the compound employed, the mode of administration, the desired treatment, and the indicated disorder. For example, the daily dosage of the compounds of the present disclosure, when inhaled, can range from 0.05 micrograms per kilogram of body weight (μg / kg) to 100 micrograms per kilogram of body weight (μg / kg). Alternatively, when the compounds are administered orally, the daily dosage of the compounds of the present disclosure can range from 0.01 micrograms per kilogram of body weight (μg / kg) to 100 milligrams per kilogram of body weight (mg / kg).

[0078] The protein having the amino acid sequence of SEQ ID NO: 1 that is PEGylated to form the drug substance described herein can be used as is, but is usually administered in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.Therefore, the present disclosure further provides a pharmaceutical composition comprising the drug substance described herein in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0079] Pharmaceutically acceptable adjuvants, diluents, or carriers that may be used in the pharmaceutical compositions of the present disclosure are those conventionally employed in the field of pharmaceutical formulations, and include, but are not limited to, sugars, sugar alcohols, starches, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerin, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts or electrolytes such as zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat (lanolin).

[0080] The pharmaceutical compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, enterally, nasally, bucally, vaginally, or via an implanted reservoir. In one embodiment, the pharmaceutical composition may be administered orally. In one embodiment, the pharmaceutical composition may be administered subcutaneously. The pharmaceutical compositions of the present disclosure may contain any conventional non-toxic pharmaceutically acceptable adjuvant, diluent, or carrier. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0081] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. Suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween® 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable adjuvant, diluent, or carrier, for example, as a solution in 1,3-butanediol. Among the acceptable adjuvants, diluents, and carriers that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly used as solvents or suspending media. For this purpose, any bland, fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.

[0082] The pharmaceutical compositions of the present disclosure can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, powders, granules, and aqueous suspensions and solutions.These dosage forms are prepared according to techniques well known in the field of pharmaceutical formulation.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dried cornstarch.For oral administration of aqueous suspensions, the active ingredient is combined with emulsifying and suspending agents.If desired, certain sweeteners and / or flavorings and / or colorings may be added.

[0083] The pharmaceutical composition of the present disclosure can also be formulated in the form of suppositories for rectal administration.These compositions can be prepared by mixing active ingredient with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature, so that they melt in the rectum and release active ingredient.Such materials include but are not limited to cocoa butter, beeswax and polyethylene glycol.

[0084] The pharmaceutical compositions of the present disclosure can be administered by nasal aerosol or inhalation. Such compositions can be prepared according to techniques well known in the art of pharmaceutical formulation, and can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0085] The pharmaceutical compositions herein may be in a form for administration through the circulatory system, as described in International Publication Nos. 2015 / 153102, 2016 / 183482, and 2018 / 009838, each of which is incorporated herein by reference in its entirety. The CBS protein may be encoded by a recombinant nucleic acid expressed by enucleated hematopoietic cells (EHCs), including erythroid cells or thrombus-like cells. For example, the erythroid cells are red blood cells, erythrocytes, or reticulocytes. For example, the thrombus-like cells are platelets. In certain embodiments, the encoded CBS protein is fused to a translated membrane-anchored polypeptide. In certain embodiments, the CBS protein is localized on the surface of the EHC. The CBS protein may be cleaved in the extracellular space to activate the enzyme. Alternatively, the internally localized CBS protein may be released into the extracellular space upon lysis of the EHC. Alternatively, the enzymatic target of the CBS protein enters the EHC and then exits through the membrane after modification. In certain embodiments, the CBS protein has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 of the Sequence Listing, or SEQ ID NO: 2, 3, or 13 of WO 2017 / 083327 (incorporated herein in its entirety).

[0086] Depending on the mode of administration, the pharmaceutical composition contains from 0.05 to 99% w (weight percent), more specifically from 0.05 to 80% w, even more specifically from 0.10 to 70% w, and even more specifically from 0.10 to 50% w of the active ingredient (all weight percentages based on the total composition).

[0087] Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described, for example, in "Pharmaceutics - The Science of Dosage Form Design", M. E. Aulton, Churchill Livingstone, 1988, which is incorporated herein by reference in its entirety.

[0088] A buffer solution of disodium hydrogen phosphate (dihydrate), sodium chloride, potassium chloride, and potassium dihydrogen phosphate is introduced into the drug substance through diafiltration. The drug is formulated at a target concentration of 20-30 mg / mL, or approximately 25 mg / mL, in PBS (pH = 7.4 ± 0.2). The PBS buffer contains disodium hydrogen phosphate (dihydrate) (11.4 mM), sodium chloride (137 mM), potassium chloride (2.7 mM), and potassium dihydrogen phosphate (1.98 mM). Table 1 provides formulation details.

[0089] [Table 1]

[0090] Table 2 provides unit dose compositions of examples of the pharmaceutical products described herein.

[0091] [Table 2]

[0092] In certain embodiments, the pharmaceutical is formulated for an exposure of about 50 mU / μL in a subject. The lyophilized formulation can be used for administration to humans upon reconstitution. A.Lyophilization The pharmaceutical composition may be a lyophilized formulation. In some embodiments, the lyophilized formulation comprises a drug substance, a buffer, and an excipient. In certain embodiments, upon reconstitution of the lyophilized formulation in a suitable reconstitution buffer, water, or any other pharmaceutically acceptable adjuvant, diluent, or carrier, the concentration of the drug substance is between about 20 and 30 mg / ml. In some embodiments, the concentration of the drug substance is about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, or about 30 mg / ml. In some embodiments, the concentration of the drug substance is about 25.4 mg / ml. In certain embodiments, upon reconstitution of the lyophilized formulation in a suitable reconstitution buffer, water, or any other pharmaceutically acceptable adjuvant, diluent, or carrier, the buffer is potassium phosphate at a concentration of 15 mM. In certain embodiments, the excipient is trehalose at a concentration of 8% (w / v). In some embodiments, the formulation comprises sucrose such that upon reconstitution of the lyophilized formulation in an appropriate reconstitution buffer, water, or a pharmaceutically acceptable adjuvant, diluent, or carrier, the concentration of sucrose is 5%. In some embodiments, the collapse onset temperature (Tc,on) is −21° C. as determined by freeze-drying microscopy. In some embodiments, the formulation has a pH of 7.5.

[0093] In some embodiments, the lyophilization process can be carried out in 48 hours or less without melting of the crystalline cake structure. The lyophilization process can be optimized to adjust one or more of the following parameters or properties, such as, but not limited to, (i) reduced reconstitution time of the lyophilized formulation (e.g., less than 1 minute), (ii) reduced viscosity allowing for more concentrated pharmaceutical products, (iii) incorporation of an isotonic buffer to minimize pain to the patient, and / or (iv) reduced dePEGylation.

[0094] Lyophilized formulations can be prepared using the following protocol. Three days prior to formulation preparation, 20-30 mg / ml or approximately 25 mg / ml of drug substance (stored at -80°C) is thawed in a refrigerator at 2-8°C for 72 hours. After thawing, the drug substance is homogenized by gentle swirling. Dialysis is performed under controlled conditions at 2-8°C for 24 hours. Using a dialysis cassette with a 20 kDa cutoff, the buffer is exchanged three times, each time at a volume ratio of 1:50 or greater. The buffer is exchanged after 3 and 6 hours of total dialysis time. The final dialysis step is performed overnight. After dialysis, the formulation is recovered from the dialysis cassette and filtered using a 0.22 μm polyvinylidene difluoride (PVDF) filter. After filtration, vials are filled to a 1.0 ml fill volume under laminar flow conditions.

[0095] Freeze-drying is performed in an Epsilon 2-12D pilot-scale freeze-dryer (Martin Christ, Osterode, Germany). Chamber pressure is controlled by a capacitance gauge and regulated by a vacuum pump and controlled nitrogen dosage.

[0096] After the vials are equilibrated to 5°C, they are frozen to -45°C and equilibrated at -45°C for an additional 5 hours. The shelf temperature is set to -15°C for 31 hours of primary drying. Secondary drying is carried out for 2.5 hours at a shelf temperature of 40°C. At the end of the lyophilization process, the chamber is vented with nitrogen to 800 mbar and the vials are stoppered by lifting the shelf. After stoppering, the chamber is vented with nitrogen to atmospheric pressure. Table 3 shows the lyophilization process parameters after cycle optimization.

[0097] [Table 3]

[0098] During the freeze-drying process, product temperature, shelf temperature, cooler temperature and chamber pressure (capacitance and Pirani gauges) are monitored. Product temperature is monitored by a Pt100 sensor (OMEGA™).

[0099] V. Treatment of Diseases, Disorders or Conditions Individuals with CBSDH are typically asymptomatic at birth, and unless treated, symptoms develop over time in these individuals, some as early as infancy, many in childhood, and, if it is a spectrum disorder, in some patients symptoms only manifest in adulthood (Yap, 2005; Mudd et al. Am J Hum Genet 1985;37:1-31; Morris et al. Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 2017;40:49-74; Mudd et al. Scriver CL, Beaudet AL, Sly WS, each of which is incorporated herein by reference in its entirety). WS, Valle D (eds.) The Metabolic and Molecular Basis of Genetic Diseases Inherited Diseases. 7th ed., New York: McGraw Hill; 2001; 1279-1327). Four major organ systems are typically involved: the eye, skeleton, and vascular system, as well as the CNS. Other organs, such as the liver, pancreas, gastrointestinal tract, and skin (including hair follicles), may also be involved (see Mudd et al., Am J Hum Genet 1985; 37:1-31; Non-Patent Document 12; Muacevic-Katanec et al., Coll Antropol 2011; 35:181-185; Suri et al., J Neurol Sci 2014; 347:305-309, each of which is incorporated herein by reference in its entirety).

[0100] Accumulating data indicate that a decrease in Hcy levels can serve as an indicator of successful application of enzyme therapy (ET) in CBSDH. This is consistent with the definition by the NIH-FDA Biomarker Working Group of a "pharmacodynamic / response biomarker whose levels change in response to exposure to a drug," and even more so, in the case of ET for CBSDH, a marker closely related to the mechanism of drug action (see FDA-NIH Biomarker Working Group. BEST (Biomarkers, Endpoints, and other Tools) Resource [Internet]. Silver Spring, MD: U.S. Food and Drug Administration; 2016-. Reasonably Likely Surrogate Endpoint. September 25, 2017. Co-published by the National Institutes of Health, Bethesda, MD. ncbi.nlm.nih.gov / books / NBK326791 / , which is incorporated herein by reference in its entirety). Thus, blood or plasma Hcy has long been recognized not only as a useful marker for pharmacodynamic studies but also as a "reasonably worthy surrogate endpoint" for homocystinuria (HCU).

[0101] In infants and children with CBSDH, the priority is to prevent complications associated with CBSDH and ensure appropriate growth and normal intellectual development (see Non-Patent Document 12, incorporated herein by reference in its entirety). In patients diagnosed later in life, the aim of treatment should be to prevent life-threatening thromboembolic events and minimize the progression of established complications. To address these goals, the biochemical abnormalities associated with CBSDH must be improved, and if possible, normalized (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0102] According to the 2016 Guidelines for the Diagnosis and Management of CBSDH, Hcy levels should be maintained as close to normal as possible (10–15 μmol / L or less). Because this is typically not possible in CBSDH patients with available treatments, more ambitious targets of less than 50 μmol / L for pyridoxine-responsive CBSDH patients and less than 100 μmol / L for non-pyridoxine-responsive patients have been suggested (see Non-Patent Document 12, incorporated herein by reference in its entirety). As previously mentioned, non-pyridoxine-responsive patients tend to have higher Hcy levels than pyridoxine-responsive patients. While two targets are recommended for patients with the same disease, these targets are designed to be achievable rather than optimal in order to minimize complications.

[0103] Overall, the effectiveness of long-term treatments required to manage CBSDH is subject to insufficient or inconsistent lifelong adherence, particularly because they most frequently rely on dietary restriction and supplementation. ET for CBSDH may avoid many of these pitfalls. By compensating for the metabolic deficiency in CBSDH through a mechanism that does not require severe Met restriction or Cys supplementation, ET is expected to achieve more consistent Hcy reduction without dangerously elevating Met levels and while allowing for dietary liberalization or normalization.

[0104] Although there is currently no cure for CBSDH that corrects the underlying genetic cause of the condition, a commonly accepted treatment goal is to reduce tHcy levels as much as possible (see Non-Patent Document 12, incorporated herein by reference in its entirety). As a result, current treatments are directed at correcting the biochemical abnormality, thereby reducing the risk of adverse clinical manifestations of the disease. Hcy levels are rarely completely normalized by currently available treatments for CBSDH patients.

[0105] In most patients, a combination of strategies is required to achieve treatment targets. These include: 1) increasing residual CBS activity by administering pharmacological doses of pyridoxine (together with folic acid, vitamin B6, a cofactor of CBS) to pyridoxine-sensitive patients (see Yap et al., Arterioscler Thromb Vasc Biol. 2001 December;21(12):2080-5, incorporated herein by reference in its entirety); 2) reducing methionine load through severe dietary / protein restriction while supplementing the diet with products beyond metabolic blockade; and 3) enhancing alternative metabolic pathways to counteract the effects of CBS deficiency, e.g., administering betaine (a methyl donor) to enhance remethylation of Hcy to Met. In certain embodiments, folic acid supplementation and (if needed) vitamin B12 supplementation are provided (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0106] A. Current Treatments for CBSDH In some embodiments, patients with CBSDH receive adequate folic acid supplementation and (if necessary) vitamin B 12 (See, e.g., J. Med. Chem. 1999, 14:101-102, incorporated herein by reference in its entirety.) In addition, patients should be treated with pyridoxine therapy (if responsive), Met restriction, Cys-replete diet, and / or betaine therapy. In most patients, a combination of strategies is required to achieve treatment targets.

[0107] The most commonly prescribed treatment was a combination of diet and betaine, followed by betaine alone and diet alone (see Adam et al. Mol Genet Metab 2013;110:454-459, incorporated herein by reference in its entirety). Once patients were over 16 years of age, these patients were often prescribed betaine alone, without diet, in recognition of the lack of adult patient compliance with Met-restricted diets. However, compliance with betaine in adults was also lacking. Median protein intake varied widely among patients and increased dramatically with age.

[0108] Consistent with these findings, a recent report noted that only 4 adult patients out of 24 patients prescribed a low-protein diet with specific CBSDH-appropriate amino acid supplementation complied with the treatment (see Lorenzini et al., J Inherit Metab Dis. 2017 Oct. 4, incorporated herein by reference in its entirety). CBSDH experts have noted similarly wide variability in the United States (Orphan Technology Scientific Advice Board, a group of physicians who are U.S. CBSDH experts). A study comparing tHcy levels in untreated patients with those in treated patients (25 and 93 patients, respectively) concluded that there was no significant difference between the two groups (tHcy ranged from 15.7 to 281.4 and 4.8 to 312 μmol / L, respectively; medians were 125.0 and 119.0 μmol / L, respectively), but the study did not provide details about the patients' treatment regimens (Stabler et al., 2004, incorporated herein by reference in its entirety). (See JIMD Rep 2013;11:149-163.) These results suggest all or a combination of the following conclusions: patients had a heterogeneous presentation of disease, standard treatments were ineffective, and / or treatment adherence was poor.

[0109] 1. Pyridoxine At the time of diagnosis, patients are tested for responsiveness to pyridoxine, a cofactor of CBS. Administration of pharmacological doses of pyridoxine (vitamin B6) increases the residual activity of CBS in individuals who are shown to be pyridoxine responsive. While the definition of pyridoxine responsiveness varies widely from site to site, the 2016 guidelines, written as part of the European Network and Registry for Homocystinuria and Methylation Defects (EHOD), defined pyridoxine responsiveness as a 20% decrease in tHcy levels within 6 weeks of pyridoxine exposure. Patients with severely elevated tHcy levels and those with mildly or moderately elevated tHcy levels may both be defined as responsive, despite presenting with very different tHcy levels. Furthermore, different treatment centers define pyridoxine responsiveness differently; therefore, categorizing patients by tHcy levels rather than pyridoxine responsiveness is more accurate and precise. In general, patients who respond to pyridoxine have some residual CBS activity and therefore lower tHcy levels, resulting in less severe presentations.

[0110] Pyridoxine is generally considered safe in patients with CBSDH (see Non-Patent Document 8, incorporated herein by reference in its entirety). Its most commonly reported side effects include peripheral neuropathy in patients treated with high doses, defined as greater than 900 mg / day (see Schaumburg et al. N Engl J Med 1983;309:445-448; Ludolph et al. Eur J Pediatr 1993;152:271, which are incorporated herein by reference in their entireties), apnea and unresponsiveness in neonates receiving 500 mg / day of pyridoxine (see Mudd et al. Am J Hum Genet 1985;37:1-31, which are incorporated herein by reference in their entireties), and rhabdomyolysis (see Shoji et al. J Inherit Metab Dis 1998;21:439-440, which are incorporated herein by reference in their entireties).

[0111] Although pyridoxine treatment is widely used, it provides only a modest reduction in tHcy levels, and because their starting levels are many times above normal, most patients defined as responsive are unable to significantly reduce, much less normalize, tHcy levels with pyridoxine alone (see Non-Patent Document 12, incorporated herein by reference in its entirety). In addition to long-term pyridoxine treatment, pyridoxine-responsive CBSDH patients are recommended to also receive folic acid and, if needed, vitamin B12 supplementation.

[0112] 2. Dietary restrictions Lifelong dietary restriction has previously been recommended for all CBSDH patients (see, e.g., Walter et al., Eur J Pediatr. 1998 April; 157 Suppl 2:S71-6, incorporated herein by reference in its entirety). The recommended diet is extremely restrictive and aims to reduce Met intake by restricting protein content.

[0113] The current mainstay of treatment for patients with CBSDH is a lifelong low-protein diet containing only 5 g of natural protein per day, supplemented with Met-free L-amino acids (www.hcunetworkamerica.org), often with additional Cys (Non-Patent Document 6; Non-Patent Document 12, incorporated herein by reference in their entirety). Severely restrictive diets consist of low-methionine grain diets, low-methionine fruits and vegetables, low-methionine medical foods, oils, and sugars. Foods containing moderate amounts of Met, such as meat, poultry, fish, eggs, milk, yogurt, cheese, soy products, nuts, legumes, and many fruits and vegetables, should be avoided. Because natural CBS is a key enzyme in Met metabolism, ingestion of Met, an essential amino acid found in many foods, results in elevated plasma concentrations of tHcy and decreased concentrations of the downstream metabolites Cth and Cys.

[0114] Prepared meals, baked goods, and packaged foods often contain milk, eggs, or wheat flour and must be highly restricted. The amount of protein required and tolerated by each CBSDH patient varies and may fluctuate over time. This amount is adjusted according to tHcy levels, which are monitored with frequent blood tests (ASIEM Low Protein Handbook for Homocystinuria). Most patients receiving dietary treatment also require daily consumption of an unpalatable, Met-free synthetic amino acid formula to prevent secondary malnutrition and for adequate growth in children and nutrition in adults (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0115] Although dietary modification combined with vitamin supplementation can reduce tHcy levels to some extent in individuals who adhere well to a highly restrictive diet, tHcy levels in most patients with CBSDH remain several times to several orders of magnitude higher than normal. For most individuals, achieving full lifelong adherence to dietary modification is highly challenging, and the resulting periods of poor metabolic control have cumulative deleterious effects (see, e.g., J. Pediatr. 1998, April; 157 Suppl. 2:S71-6; J. Pediatr. 1998, April; 157 Suppl. 2:S71-6; J. Pediatr. 1998, April; 157 Suppl. 2:S71-6; J. Pediatr. 1998, November; 4(8):557-62, all of which are incorporated herein by reference). Dietary adherence is often poor and typically worsens during adolescence and adulthood (see, e.g., Walter et al., Eur J Pediatr. 1998, April; 157 Suppl. 2:S71-6; J. Pediatr. 1998, April; 157 Suppl. 2:S71-6; J. Pediatr. 1998, November ... Furthermore, eating high-Met foods does not induce an immediate negative physical reaction, making dietary compliance even more difficult (www.hcunetworkamerica.org). Met restriction is even more difficult in children because of the need to ensure sufficient Met to facilitate growth and development. The majority of patients undergoing dietary treatment also require Cys-rich, Met-free L-amino acid supplements for adequate growth in children and for adequate nutrition in adults (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0116] A recent report noted that only 4 adult patients out of 24 patients prescribed a low-protein diet with specific CBSDH-appropriate amino acid supplementation complied with the treatment (Lorenzini et al., 2006, incorporated herein by reference in its entirety). (See J Inherit Metab Dis. (2018) 41:109-115.) Several CBSDH experts have described similarly wide variability in the United States.

[0117] 3. Betaine Supplementation Problems associated with heavily diet-based treatments have necessitated other approaches to lowering Hcy, most notably betaine (N,N,N-trimethylglycine, commercially available as CYSTADANE™) supplementation administered at least twice daily. Betaine is rarely effective as monotherapy (Sakamoto et al., Pediatr Int 2003;45:333-338, incorporated herein in its entirety) and is usually used as an adjunct to a pyridoxine- and / or Met-restricted diet (see Non-Patent Document 12, incorporated herein in its entirety).

[0118] Rather than addressing the underlying CBS defect, betaine induces an alternative pathway, resulting in remethylation of Hcy to Met, correcting the partial misfolding of CBS mutants (Kopecka et al., J. Med. 2004, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 1 Inherit Metab Dis 2011;34:39-48. In the presence of betaine, the enzyme betaine homocysteine ​​methyltransferase (BHMT) remethylates Hcy to Met (Singh et al., incorporated herein in its entirety). (Et al.) Genet Med 2004;6:90-95), thus partially lowering Hcy levels while increasing already highly elevated Met levels. Downstream metabolites of CBS are not improved by betaine administration, and Cys supplementation may be required. Furthermore, betaine treatment has been shown to reduce acute cerebral edema with (Devlin et al. J Pediatr 2004;144:545-548; Yaghmai et al. Am J Med Genet 2002;108:57-63, all of which are incorporated herein by reference) and without (Vatanavicharn et al. J Inherit Metab Dis 2008;31 Suppl 3:477-481; Brenton et al. J Child Neurol 2014;29:88-92; Sasai et al. Tohoku J Exp Med 2015;237:323-327) have been associated with cerebral white matter abnormalities—signs of vascular damage in the brain (Prins et al. Nat Rev Neurol 2015;11:157-165, incorporated herein by reference in its entirety).

[0119] Betaine has an unpleasant taste (Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2:S71-6, incorporated herein in its entirety) and can result in an unpleasant fishy body odor and / or breath (see Manning et al. JIMD Rep 2012;5:71-75, incorporated herein in its entirety), both effects potentially exacerbated by the requirement for high doses (greater than 6 g / day in adult and pediatric patients). As a result, compliance is commonly poor (see Adam et al. Mol Genet Metab. 2013 Dec;110(4):454-9; Walter et al. Eur J Pediatr. 1998 Apr;157 Suppl 2:S71-6; Sakamoto et al. Pediatr Int 2003;45:333-338, which are incorporated herein by reference in their entireties).

[0120] A pharmaceutical formulation of betaine, CYTADANE™, was approved by the FDA in 2006 and is indicated to lower elevated blood Hcy in homocystinuric disorders, including CBS deficiency, 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency, and cobalamin cofactor metabolism (cbl) deficiency (Recordati. CYTADANE, incorporated herein by reference in its entirety). TM Product Information(PI).2017.Ref Type:Online source reference).

[0121] The largest survey to date of dietary habits among CBSDH pyridoxine-nonresponsive patients has indicated that betaine has become a popular treatment option, especially in late-diagnosed patients, adolescents, and adults. Because there are no controlled studies examining the long-term efficacy of betaine when administered without a meal, the use of betaine as primary therapy in 34% of patients without a meal is likely due to a lack of dietary adherence (Adam et al., 2004, incorporated herein by reference in its entirety). (See, e.g., et al.) Mol Genet Metab 2013;110:454-459.) Indeed, a study in the CBSDH mouse model found that the ability of betaine treatment to significantly lower tHcy decreased over time (Maclean KN. Betaine Treatment of Cystathionine Beta-Synthase Deficiency Homocystinuria: Will It Work and Will It Improve?, incorporated herein by reference in its entirety). treatment of cystathionine b-synthase-deficient homocystinuria;does it work and (see Dove press 2012;2:23-33).

[0122] 4. Antiplatelet therapy In addition to Hcy-lowering therapy, patients with poorly controlled Hcy levels and / or those with additional risk factors for thrombosis (e.g., factor V Leiden, previous thrombosis, and pregnancy) may benefit from treatment with antiplatelet agents (e.g., aspirin, dipyridamole, or clopidogrel) (see Non-Patent Document 12, incorporated herein by reference in its entirety). COUMADIN™ blood thinners may also be used in patients with previous venous thrombosis. However, anticoagulants are associated with an increased risk of cerebral hemorrhage, and their use should be determined on an individual basis (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0123] 5. Clinical outcomes with current treatment The manifestations of CBSDH continue to progress with classic clinical symptoms culminating in varying degrees of disability, affecting the quality of life of affected individuals. Regardless of the individual's age at onset, loss of biochemical control at any age is associated with the development of serious, potentially life-threatening complications (see Walter et al., Eur J Pediatr. 1998 April;157 Suppl 2:S71-6, incorporated herein by reference in its entirety).

[0124] Treatment must continue throughout life, as periods of poor metabolic control have cumulative adverse effects that can lead to severe complications and premature death (see Non-Patent Document 12, incorporated herein by reference in its entirety).

[0125] Without treatment, pyridoxine-unresponsive CBSDH has a poor prognosis, with a significantly reduced life expectancy. Since CBSDH was first described in 1962, no randomized controlled trials of dietary or other treatments for CBSDH have been performed (see Carson et al., Arch Dis Child. 1969 June;44(235):387-92; Gerritsen et al., Biochem Biophys Res Commun. 1962 December 19;9:493-6, which are incorporated herein by reference in their entireties). However, only a few observational studies have been published.

[0126] An international study documenting the natural history of 629 untreated CBS patients showed that the risk of complications increased with age (see Mudd et al. Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety). Treatment (pyridoxine, Met-restricted diet) was observed to reduce plasma tHcy levels and significantly reduced the risk of thromboembolic events and lens dislocation, but poor adherence to the restricted diet was observed.

[0127] Yap et al. (Arterioscler Thromb Vasc Biol. 2001 Dec;21(12):2080-5) conducted an international multicenter study in 158 treated patients. The incidence of cardiovascular events was significantly reduced in this treatment group compared with historical control data from Mudd et al. (Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety). This apparent benefit correlated with lower (but not normalized) tHcy plasma levels in treated patients. It was noted that consistent adherence to the required regimen was very difficult.

[0128] Overall, although several CBSDH treatment strategies are available, they fail to restore near-normal tHcy levels in most patients. Furthermore, their long-term efficacy is subject to poor or inconsistent lifelong adherence. Thus, consistent Hcy reduction is difficult to maintain in CBSDH patients. The pharmaceutical treatments described herein aim to avoid many of these pitfalls. By compensating for the metabolic defects in CBSDH through a mechanism that requires neither severe Met restriction nor Cys replacement, enzyme therapy (ET) treatment is expected to achieve more consistent Hcy reduction without dangerously elevating Met levels.

[0129] VI. Dosage and Administration In certain embodiments, the pharmaceutical agent can be administered to the subject by subcutaneous (SC), intravenous (IV) or intraperitoneal (IP) injection.In one embodiment, the pharmaceutical agent can be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times.In another embodiment, the pharmaceutical agent is administered more than 20 times.In another embodiment, the pharmaceutical agent is administered more than 100 times.Alternatively, the pharmaceutical agent can be administered for the remaining life of the subject.

[0130] In certain embodiments, the administration of the pharmaceutical agent can be repeated every 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, and every month. In certain embodiments, the administration of the pharmaceutical agent is performed every 3 days, every 2 days, or once a day.

[0131] In certain embodiments, the administration of the pharmaceutical agent can be a series of administrations separated by minutes, hours, days, or weeks. For example, the number of series of administrations can be one, two, three, four, five, or six. As a non-limiting example, the subject is administered three doses 24 hours apart. As another non-limiting example, the subject is administered five doses 12 hours apart. The subject can be a human.

[0132] In some embodiments, the administration of pharmaceuticals can be according to a dosing schedule of a series of administrations, with a gap between the first and second series of administrations.The gap between administrations can be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months.The number of series of administrations can be 2, 3, 4, 5, or 6 times. As a non-limiting example, a subject is administered a first series of five doses 12 hours apart, and then 14 days after the first dose, the subject is administered a second series of five doses 12 hours apart. As another non-limiting example, a subject is administered two series of doses over an eight-week period, with the first series being one dose twice per week for two weeks and the second series being three doses per week for six weeks.

[0133] In certain embodiments, a pharmaceutical agent can be administered at least once after betaine is administered to a subject.The time between betaine administration and pharmaceutical agent administration can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, a quarter, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months.As a non-limiting example, a pharmaceutical agent can be administered 14 days after betaine is administered to a subject. As another non-limiting example, the subject may be administered betaine followed by a second administration of the pharmaceutical agent. As another non-limiting example, the pharmaceutical agent may be administered 14 or 15 days after the administration of betaine.

[0134] In certain embodiments, a pharmaceutical agent can be administered to a subject in combination with betaine. The combination can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 times. In certain embodiments, a pharmaceutical agent can be administered in combination with betaine more than 15 times. Additional combination therapies that can be administered to a patient include a pharmaceutical agent and at least one treatment to reduce tHcy levels, such as a very low protein / Met diet and / or vitamin / supplement.

[0135] In one embodiment, the dose of the pharmaceutical agent administered to a subject can be between about 0.25 mg / kg and about 10 mg / kg. For example, the dose can be one of about 0.33 mg / kg, about 0.66 mg / kg, 1.0 mg / kg, or 1.5 mg / kg. Alternatively, the dose can be about 2 mg / kg, about 7 mg / kg, and about 10 mg / kg. For example, the dose can be about 0.5 mg / kg. Alternatively, the therapeutically effective amount is a dose selected from the ranges of about 5.0 mg / kg to about 50 mg / kg and about 10.0 mg / kg to about 25 mg / kg. For example, dosages may be about 0.25 mg / kg, about 0.33 mg / kg, about 0.66 mg / kg, about 1.00 mg / kg, about 1.10 mg / kg, about 1.20 mg / kg, about 1.30 mg / kg, about 1.40 mg / kg, about 1.50 mg / kg, about 1.60 mg / kg, about 1.70 mg / kg, about 1.80 mg / kg, about 1.90 mg / kg, about 2.00 mg / kg, about 3.00 mg / kg, about 4.00 mg / kg, about 5.00 mg / kg, about 6.00 mg / kg, about 7.00 mg / kg, about 8.00 mg / kg, about 9.00 mg / kg, about 10.00 mg / kg, about 11.00 mg / kg, about 12.00 mg / kg, about 13.00 mg / kg, about 14.00 mg / kg, about 15.00 mg / kg, about 16.00 mg / kg, about 17.00 mg / kg, about 18.00 mg / kg, about 19.00 mg / kg, about 20.00 mg / kg, about 21.00 mg / kg, about 22.00 mg / kg, about 23.00 mg / kg, about 24.00 mg / kg, about 25.00 mg / kg, about 26.00 mg / kg, about 27.00 mg / kg, about 28.00 mg / kg, about 29.00 mg / kg, about 30.00 mg / kg, about 31.00 mg / kg, about 32.00 mg / kg, about 33.00 mg / kg, about 34.00 mg / kg, about 35.00 mg / kg, about 36.00 mg .00mg / kg, about 5.00mg / kg, about 6.00mg / kg, about 7.00mg / kg, about 8.00mg / kg, about 9.00mg / kg, about 10.0mg / kg, about 11.0mg / kg, about 1 2.0mg / kg, about 13.0mg / kg, about 14.0mg / kg, about 15.0mg / kg, about 16.0mg / kg, about 17.0mg / kg, about 18.0mg / kg, about 19.0mg / kg, about 20 0.0mg / kg, about 21.0mg / kg, about 22.0mg / kg, about 23.0mg / kg, about 24.0mg / kg, about 25.0mg / kg, about 26.0mg / kg, about 27.0mg / kg, about 28.0mg / kg, about 29.0mg / kg, about 30.0mg / kg, about 31.0mg / kg, about 32.0mg / kg, about 33.0mg / kg, about 34.0mg / kg, about 35.0mg / kg, about 36 0.0 mg / kg, about 37.0 mg / kg, about 38.0 mg / kg, about 39.0 mg / kg, about 40.0 mg / kg, about 41.0 mg / kg, about 42.0 mg / kg, about 43.0 mg / kg, about 44.0 mg / kg, about 45.0 mg / kg, about 46.0 mg / kg, about 47.0 mg / kg, about 48.0 mg / kg, about 49.0 mg / kg, and about 50.0 mg / kg.

[0136] In certain embodiments, the medicament is administered to a subject on a methionine-restricted diet. Alternatively, the medicament is administered to a subject not on a methionine-restricted diet. In certain embodiments, the pharmaceutical agent can be co-administered with another therapeutic agent for treating CBSDH. As used herein, "co-administered" refers to the administration of two or more components. These components for co-administration include, but are not limited to, betaine or vitamin B6. Co-administration can be simultaneous or with a time lapse between administrations, e.g., 1 second, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, It refers to the administration of two or more components over 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 1.5 days, 2 days, or 3 days. In certain embodiments, the time lapse between the administration of two or more components is more than 3 days.

[0137] In certain embodiments, the pharmaceutical agent may be administered to a patient chronically via subcutaneous (SC) injection with an initial administration interval of once a week. For example, the drug may be administered weekly for six doses. In certain embodiments, the subject may be within the age range of 18 to 65 years. In certain embodiments, subjects as young as 16 years old may be similarly treated.

[0138] In certain embodiments, administration occurs over 1, 2, 3, 4, 5, or 6 days. In certain embodiments, administration occurs over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks.

[0139] In certain embodiments, the pharmaceutical agent is administered as a combination therapy with pyridoxine (also known as vitamin B6) and / or antiplatelet therapy. VII. Patient stratification In certain embodiments, individuals eligible for effective enzyme therapy using the pharmaceutical products described herein include patients who have been diagnosed with CBSDH based on confirmation of hereditary CBS deficiency homocystinuria by mutation analysis of the CBS gene and plasma tHcy levels of 80 μM or greater.

[0140] A. Clinical Presentation Guidelines for the diagnosis and management of CBSDH suggest that the disease should be suspected in children who present with severe or rapidly progressive myopathy, lens dislocation, and / or developmental delay (see Non-Patent Document 12, incorporated herein by reference in its entirety). Testing is also warranted in adults who present with thromboembolic disease and / or lens dislocation but no other symptoms, as well as in adults with multisystem disorders involving ocular, connective tissue, neuropsychiatric, and vascular complications (see Mudd et al., Am J Hum Genet 1985;37:1-31; Non-Patent Document 12; Kelly et al., Neurology 2003;60:275-279, each incorporated herein by reference in its entirety).

[0141] B. Biochemical analysis Plasma tHcy levels are determined using the sum of all free and bound homocysteine ​​species after treating plasma with a reducing agent. In healthy individuals with stable dietary habits, tHcy levels remain relatively constant over time (see, e.g., J. Med. Chem. 2001;47:1430-1436; McKinley et al., Clin. Chem. 2001;47:1430-1436, each of which is incorporated herein by reference in its entirety). However, consumption of a protein-rich meal can increase tHcy levels by approximately 10% over a period of several hours (see, e.g., Verhoef et al., Am. Chem. 2001;47:1430-1436, each of which is incorporated herein by reference in its entirety). (See J Clin Nutr 2005;82:553-558.) A study in individuals with hyperhomocysteinemia (tHcy >40 μmol / L) found that intra-individual tHcy levels varied by up to 25% over a 4-8 month period. However, no information was provided about variability in diet, assay method, and time of sampling, which hindered the ability to interpret the data (See Non-Patent Document 11, incorporated herein by reference in its entirety).

[0142] In populations not receiving folic acid supplementation, the corresponding upper reference limits are approximately 15 and 20 μmol / L, respectively. To support a diagnosis of CBS in newborns, plasma tHcy is expected to be between 50 and 100 μmol / L and plasma Met is expected to be between 200 and 1500 μmol / L (i.e., 3-23 mg / dL) (see Non-Patent Document 2, incorporated herein by reference in its entirety). To support a diagnosis of CBS in untreated older individuals, plasma tHcy is expected to be greater than 100 μmol / L and plasma Met is expected to be greater than 50 μmol / L (i.e., greater than 0.7 mg / dL). Control newborns or older individuals are expected to have plasma tHcy less than 15 μmol / L and Met between 10 and 40 μmol / L (0.2-0.6 mg / dL).

[0143] High-to-high-normal Met levels (references typically range from 40-45 μmol / L and 12-15 μmol / L, respectively) combined with low-to-low-normal Cth levels (references range from 0.05-0.08 μmol / L and 0.35-0.5 μmol / L, respectively) can be useful in distinguishing CBSDH from HCU caused by genetic and nutritional disorders of Hcy methylation (see Non-Patent Document 12; Stabler et al. JIMD Rep 2013;11:149-163; Bartl et al. Clin Chim Acta 2014;437:211-217, each of which is incorporated herein by reference in its entirety). Another useful test uses radioactive or deuterium-labeled substances to determine Cth production from Hcy and serine in cultured fibroblasts (see Non-Patent Document 12; Kraus JP. Methods Enzymol 1987;143:388-394; Smith et al. J Chromatogr B Analyt Technol Biomed Life Sci 2012;911:186-191, each of which is incorporated herein by reference in its entirety). However, enzyme analysis cannot always distinguish between pyridoxine-responsive and non-responsive individuals, and enzyme activity may be normal in mild cases (see Alcaide et al. Clin Chim Acta 2015;438:261-265, each of which is incorporated herein by reference in its entirety). More recently, a rapid, stable isotope assay measuring the activity of CBS released from organs into plasma was found to be 100% sensitive in pyridoxine non-responders but only 86% sensitive in pyridoxine responders (see Alcaide et al. Clin Chim Acta 2015;438:261-265; Krijt et al. J Inherit Metab Dis 2011;34:49-55, both of which are incorporated herein by reference in their entireties).

[0144] C. Molecular Diagnostics Molecular genetic testing—the gold standard diagnostic test for CBSDH—can be performed with single-gene testing or using a multi-gene panel (see Yap S., Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [Regular Online] 2005; Sacharow SJ, Picker JD, Levy HL. Homocystinuria Caused by Cystathionine Beta-Synthase Deficiency. GeneReviews 2017; Non-Patent Document 12; Katsanis et al., Nat Rev Genet 2013;14:415-426; each of which is incorporated herein by reference in its entirety). Individuals at high risk for specific CBS mutations should be screened using targeted single-gene testing. However, this is only useful in selected populations with common CBS mutations (e.g., 93% of individuals with CBSDH in Qatar carry the p.Arg336Cys;c.1006C>T mutation) and in individuals from families with known pathogenic variants. In other patients, the CBS gene can be sequenced, and gene-targeted deletion / duplication analysis can be performed only if a single pathogenic variant is found, or if no pathogenic variants are found. Alternatively, simultaneous molecular testing of multiple genes can be performed using a multi-gene panel. Methods used may include sequence analysis, deletion / duplication analysis, and other non-sequencing-based tests (see Non-Patent Document 12, incorporated herein by reference in its entirety). Typically, molecular genetic testing is reserved for high-risk populations with a limited number of prevalent mutations (see, e.g., J. Med. 2015; Huemer et al. J. Inherit. Metab. Dis. 2015;38:1007-101, each of which is incorporated herein by reference in its entirety).

[0145] D. Pyridoxine responsiveness test Pyridoxine responsiveness testing is used in clinics to determine whether patients with CBSDH should be prescribed pyridoxine supplementation. Because different treatment centers define pyridoxine responsiveness differently (see, e.g., J. Med. 2001, 144:101-102, incorporated herein by reference in its entirety), categorizing patients by tHcy levels rather than pyridoxine responsiveness is more rigorous. Pyridoxine responsiveness is not a measure of metabolic control, but rather an indicator of some residual CBS activity remaining.

[0146] E. Newborn Screening (NBS) Generally, NBS testing for CBSDH deficiency is accomplished by analyzing dried blood spots to determine Met levels. Alternatively, assessment of tHcy levels rather than Met in dried blood spots for NBS is available in a few centers worldwide. This has been adopted as a second-tier test to reduce the false-positive rate of NBS in individuals with high Met levels (Turgeon et al., 2004, incorporated herein by reference in its entirety). et al.) Clin Chem 2010;56:1686-1695), are not used to improve sensitivity or reduce false negative rates.

[0147] VIII. Phenotypic Outcome Retrospective studies have demonstrated a proportional relationship between tHcy levels and outcome: patients (treated or untreated) with the highest tHcy levels present with more severe symptoms earlier in life, while patients with lower tHcy levels present with fewer symptoms and progress less rapidly (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [regular online] 2005; Mudd et al. Am J Hum Genet 1985;37:1-31, each incorporated herein by reference in its entirety). Individuals with elevated tHcy levels typically present with growth retardation, thromboembolic events, severe myopia with posterior dislocation of the eye lens, osteoporotic fractures, Marfanoid diathesis (especially elongation of long bones), and / or psychiatric abnormalities such as learning difficulties (see Yap S., Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [Regular Online] 2005; Non-Patent Document 12; NORD, Kraus J. P., Homocystinuria due to cystathionine beta-synthase deficiency. NORD [Regular Online] 2017, each of which is incorporated herein by reference in its entirety). Reflecting the spectrum of CBS deficiency, some patients have severe childhood-onset multisystem disease, while those with less severely elevated Hcy may remain undiagnosed until adulthood (see Non-Patent Document 12, incorporated herein by reference in its entirety). Patients with severely elevated Hcy levels have significantly reduced life expectancy, but even those with moderately elevated tHcy levels suffer from multiple negative clinical outcomes.

[0148] Significant evidence points to a causal effect of elevated tHcy levels on negative clinical outcomes in four systems commonly affected in patients with CBSDH: ophthalmic, skeletal, cardiovascular, and neurological. In the ophthalmic system, frequently observed phenotypic outcomes include ectopia lentis, iridoeconia, and myopia, while less frequently observed phenotypic outcomes include glaucoma, optic nerve atrophy, retinal degeneration, retinal detachment, cataracts, and corneal abnormalities. In the skeletal system, frequently observed phenotypic outcomes include osteoporosis, biconcave vertebrae, scoliosis, increased long bone length, irregularly widened metaphyses, metaphyseal spiculations, abnormal epiphyseal size / shape, growth arrest lines, pes cavus, and high-arched palate. Less frequently observed phenotypic outcomes include arachnodactyly, enlarged carpals, abnormal bone age, pectus carinatum / pectus excavatum, genu valgum, kyphosis, and short fourth metacarpals. In the vascular system, frequently observed phenotypic outcomes include vascular occlusion, malar flushing, and livedo reticularis. In the central nervous system, frequently observed phenotypic outcomes include mental retardation, psychiatric disorders, and extrapyramidal signs, while less frequently observed phenotypic outcomes include seizures and abnormal electroencephalograms. In additional body systems, the following phenotypic outcomes are frequently observed: pale and brittle hair, thin skin, fatty changes in the liver, inguinal hernia, myopathy, endocrine abnormalities, reduced coagulation factors, and spontaneous intestinal perforation.

[0149] Although a strong relationship between mildly elevated tHcy levels and negative outcomes has been observed, data also indicate that lowering tHcy levels positively impacts clinical manifestations. The literature on CBSDH is plagued by the rarity of the disease and subsequent smaller studies, but benefits from expanded coverage of clinical outcomes in populations with severely elevated Hcy levels. Conversely, studies in broader populations benefit from larger case numbers but smaller elevations of tHcy levels. Collectively, these studies consistently demonstrate that elevated tHcy levels strongly predict negative clinical outcomes and that pharmacological interventions to lower these levels are beneficial.

[0150] A potential outcome from treating CBSDH with the pharmaceuticals described herein is to reduce plasma tHcy concentrations to the lowest possible level while maintaining a more relaxed diet containing higher concentrations of Met and other essential amino acids than provided by other treatments for CBSDH. In infants and children with CBSDH, the priority is to prevent complications associated with CBSDH and ensure appropriate growth and normal intellectual development (see Non-Patent Document 12, incorporated herein by reference in its entirety). In patients diagnosed later in life, the priority may be to prevent life-threatening thromboembolic events and minimize the progression of established complications. To address these goals, biochemical abnormalities associated with CBSDH may be improved, and if possible, normalized (see Non-Patent Document 12, incorporated herein by reference in its entirety). A study comparing dietary management practices for CBSDH patients across 29 centers in eight European countries found that there was little consensus among treatment centers regarding the target range for plasma tHcy levels, with the median recommended target being less than 55 μM and in the range of 20 to 100 μM across the 29 centers (see Adam et al. Mol Genet Metab. 2013 Dec;110(4):454-9, incorporated herein by reference in its entirety).

[0151] Considering the 25% intra-individual variability in plasma tHcy levels tested several months apart, we avoided excluding patients with previous plasma tHcy levels of approximately 100 μM (Guttormsen et al., 2003; Guttormsen et al., 2003; Guttormsen et al., 2003; each of which is incorporated herein by reference in its entirety). A cutoff value of 80 μM or higher for tHcy levels for treatment eligibility was chosen herein to provide a level high enough to detect clinically significant declines in a small number of patients. The 25% intra-individual variability in plasma tHcy levels tested several months apart (see, e.g., [PubMed], vol. 11; Guttormsen et al., J Clin Invest. 1996; 98(9):2174-83, each of which is incorporated herein by reference in its entirety) may be due, in part, to changes in diet, medication, or supplements in CBSDH patients over time.

[0152] If left untreated, the prognosis for patients with pyridoxine-unresponsive CBSDH is bleak (see Non-Patent Document 12, which is incorporated herein by reference in its entirety). In 1985, an international retrospective study documenting the natural history of CBSDH in 629 patients with pretreatment time-to-event analysis showed that 70% of patients experienced lens dislocation by age 10 years, and 85% developed symptoms by age 12 years (Mudd et al. Am J Hum Genet 1985;37:1-31; Mudd et al., Skovby F. Disorders of transsulfuration. Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7th ed., New York: McGraw Hill; 2001; 1279-1327). Overall, 50% of affected individuals had radiographically detected spinal osteoporosis by age 15, and 23% of pyridoxine-nonresponsive patients (4% of responsive patients) died by age 30 (see Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety).

[0153] Taken as a whole, the available evidence indicates that current approaches to the treatment of CBSDH, including the use of restricted diets and dietary supplements, are ineffective in halting disease progression in most patients. As a result, there is a substantial unmet medical need to identify well-tolerated therapies that improve or normalize the metabolic abnormalities of CBSDH and slow or halt the progression of clinical manifestations of the disease.

[0154] A causal effect between elevated Hcy levels and important clinical outcomes associated with CBSDH has been observed, including ocular complications (particularly lens dislocation), skeletal outcomes (particularly osteoporosis), vascular events (particularly stroke and small vessel disease), and various CNS outcomes (particularly cognitive function). The relationship between elevated tHcy levels and negative clinical outcomes, and conversely, the relationship between decreased Hcy levels and improved clinical outcomes, has been further strengthened by multiple studies in the general population. Overall, these clinical findings emphasize the need for early CBSDH diagnosis and prompt treatment to reduce Hcy levels as close to normal as possible.

[0155] Although there are no published studies on the quality of life (QoL) of patients with CBSDH, unpublished reports indicate that patients and their caregivers suffer from the psychosocial effects of following and managing a highly restrictive and socially isolating diet and are extremely concerned about the long-term medical consequences of the disease. Not surprisingly, patients are anxious to be able to ease their diet without compromising their long-term prospects.

[0156] The strong relationship between tHcy levels and important clinical outcomes in patients with CBSDH indicates that changes in tHcy levels are a reliable surrogate marker for a combination of clinical endpoints in CBSDH. Changes in tHcy levels are therefore useful for (i) monitoring patient progression in the clinic and (ii) predicting the clinical benefit of new treatments in clinical trials.

[0157] For example, the pharmaceutical agent normalizes or increases the femoral artery flexibility of the subject compared to before administering the pharmaceutical agent to the subject.For example, I278T mice have significantly lower femoral artery flexibility compared to wild-type mice.Met-restricted diet can actually cause the femoral artery diameter of I278T mice to be smaller than that of normal diet in both mice treated with pharmaceutical agent and mice not treated with pharmaceutical agent.

[0158] Previous studies performed in three mouse models of the disease demonstrated that htCBS C15S was effective after systemic administration, as described in International Publication No. 2017 / 083327, the entire contents of which are incorporated herein by reference. These studies demonstrated up to a 90% reduction in extracellular Hcy plasma levels and intracellular Hcy levels in tissues such as the brain. Administration of the drug was observed to result in a concentration gradient, with Hcy flowing from high concentrations in the intracellular space to low concentrations in the extracellular space (where the drug can further process it). Extracellular PEGylated htCBS C15S acts as an Hcy "sink." In summary, pharmaceuticals restored regulation of the Met metabolic pathway in the CBSDH animal model.

[0159] These studies also demonstrate that SC administration of PEGylated htCBS C15S in the CBSDH mouse model corrected metabolite levels, including elevated Cth and normalized Cys levels. Additionally, PEGylated htCBS C15S positively impacted disease phenotypic manifestations in mice, including facial alopecia, liver histology, osteoporosis, body composition, diabetic retinopathy (possibly secondary to renal disease), and patchy atrophy and optic nerve atrophy due to retinal vascular occlusion or non-arteritic ischemic optic neuropathy, as well as cytokine and lipid levels. PEGylated htCBS C15S also rescued CBS knockout (KO) mice from premature death (Looker et al. Diabetologia 2003;46:766-772; Pusparajah et al. Front Physiol 2016;7:200; Gerth et al. J AAPOS 2008;12:591-596; Stanger et al. Clin Chem 2016;12:12, each of which is incorporated herein by reference in its entirety). (See Lab Med 2005;43:1020-1025; Cahill et al. Am J Ophthalmol 2003;136:1136-1150; Minniti et al. Eur J Ophthalmol 2014;24:735-743.) PEGylated htCBS C15S has also been observed to be well tolerated with no toxicological effects observed upon chronic administration in animal models of disease.

[0160] PEGylated htCBS C15S is expected to act in the extracellular space and reduce tHcy plasma concentrations regardless of patient genetics, concomitant therapy, or baseline tHcy levels. Therefore, the eligible population for the study should include both pyridoxine-responsive and non-responsive patients.

[0161] In healthy individuals, tHcy levels range from approximately 5 to 15 μM (OECD Environmental Health and Safety Publications. Series on Principles of Good Laboratory Practice and Compliance Monitoring. No. 1 ENV / MC / CHEM(98)17 Principles of Good Laboratory Practice (revised 1997), incorporated herein in its entirety), with 98% of tHcy being disulfide- or protein-bound. Only 2% of tHcy exists as an unbound, free, reduced aminothiol that can serve as a substrate for the enzyme (see EMA: Guideline on bioanalytical method validation, EMEA / CHMP / EWP / 192217 / 2009, ev. 1, July 21, 2011; ATL-15-1419 Atlanbio Study Report "LC-MS / MS determination of cystathionine-D4 as product of the cystathionine β-synthase activity in monkey plasma samples collected during the study 529736," both of which are incorporated herein by reference in their entireties). On the other hand, CBSDH patients not only present with plasma levels that can reach 400 μM or more, but also with a dramatically altered balance, with free homocysteine ​​reaching 10-25% of the tHcy value.

[0162] In mouse models, administration of PEGylated htCBS C15S resulted in a reduction in tHcy levels by up to 90%. Therefore, the initial level of free homocysteine ​​available to the enzyme (10%–25% of total) alone cannot explain the significant reduction in tHcy levels observed; an additional pool must become available to the enzyme. For example, as free Hcy becomes scarce as a result of PEG-htCBS activity, the balance between free Hcy and Hcy adducts (protein-bound or disulfide forms) in plasma shifts to favor the production of free Hcy, which can then be further processed by the enzyme.

[0163] A. Eye complications Elevated Hcy levels are a strong independent risk factor for ocular complications, particularly lens dislocation, in patients with CBSDH and in the general population. Even when prescribed pharmacological and dietary interventions, the majority of CBSDH patients ultimately develop ocular complications. Lowering Hcy levels has been shown to delay and potentially prevent lens dislocation in patients with CBSDH (Yap S., Cystathionine Beta Synthase Deficiency Homocystinuria, 2004; Yap S., 2004; Homocystinuria Due to Cystathionine Beta Synthase Deficiency ... each of which is incorporated herein by reference in its entirety). due to cystathionine beta-synthase deficiency). Orphanet Encyclopaedia [regular online] 2005; Mudd et al. Am J Hum Genet 1985;37:1-31; Martinez-Gutierrez et al. Int Ophthalmol (2011) 31:227-232; Non-Patent Document 13; Mulvihill et al. J AAPOS 2001;5:311-315; Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612; Sweetser et al. N Engl J Med 2016, 375:1879-1890; Sadiq et al. Semin Ophthalmol 2013;28:313-320; Wright et al. Homocysteine, folate, and the eye. Eye (Lond) 2008;22:989-993; Lieberman et al. Am J Ophthalmol 1966,61:252-255; Harrison et al. Ophthalmology 1998,105:1886-1890; Ramsey et al. Am J Ophthalmol 1972;74:377-385; Couser et al. Ophthalmic Genet 2017, 38:91-94; Ghorbanihaghjo et al. Mol Vis 2008, 14:1692-1697; Javadzadeh et al. Mol Vis 2010, 16:2578-2584; Seddon et al. Am J Ophthalmol 2006, 141:201-203; Coral et al. Eye(Lond) 2006, 20:203-207; Axer-Siegel et al. Am J Ophthalmol 2004, 137:84-89; Heuberger et al. Am J Clin Nutr 2002, 76:897-902; Huang et al. al.) Sci Rep 2015;5:10585; Sen et al. Indian J Clin Biochem 2008;23:255-257; Yousefi et al. Protein Pept Lett 2013;20:932-941; Gerth et al. J AAPOS 2008;12:591-596; Stanger et al. Clin Chem Lab Med 2005,43:1020-1025; Cahill et al.) Am J Ophthalmol 2003, 136:1136-1150; Minniti et al. Eur J Ophthalmol 2014, 24:735-743; Turkcu et al. Medicina (Kaunas) 2013, 49:214-218; Vessani et al. Am J Ophthalmol 2003; 136:41-46; Leibovitch et al. J Glaucoma 2003, 12:36-39; Leibovitzh et al. Medicine (Baltimore) 2016; 95:e4858; Micheal et al. Mol Vis. 2009;15:2268-2278; Clement et al. J Glaucoma 2009;18:73-78; Cumulcu et al. BMC Ophthalmol 2006;6:6; Bleich et al. J Neural Transm (Vienna) 2002;109:1499-1504; Lee et al. Curr Eye Res (See, e.g., Wang et al., Am J Ophthalmol 2004;137:401-406; Ganapathy et al., Invest Ophthalmol Vis Sci 2009;50:4460-4470). Even when prescribed pharmacological and dietary interventions, the majority of CBSDH patients ultimately develop ocular complications. Reduction of Hcy levels has been shown to delay and potentially prevent lens dislocation in CBSDH patients (see, e.g., Non-Patent Document 6, incorporated herein by reference in its entirety).

[0164] One of the most consistent and earliest manifestations of CBSDH is ectopia lentis (lens dislocation) (Mulvihill et al., 2004, incorporated herein by reference in its entirety). (See Mudd et al., J AAPOS 2001;5:311-315.) It usually occurs after age 2 years and is present by age 6 years in approximately 50% of untreated pyridoxine-nonresponsive patients and by age 10 years in 50% of untreated pyridoxine-responsive patients (See Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein in their entireties). The luxation may be partial (subluxation) or complete, may occur inferiorly or nasally, and is usually bilateral (See Mulvihill et al., J AAPOS 2001;5:311-315; Sweetser et al., N Engl J Med 2016;375:1879-1890, both incorporated herein in their entireties).

[0165] Lens dislocation is often followed by a period of rapidly progressive myopia, which can lead to significant astigmatism, monocular diplopia, and reduced best-corrected visual acuity (see Sadiq et al., Semin Ophthalmol 2013;28:313-320, incorporated herein by reference in its entirety). Overall, myopia (greater than 1 diopter [D]) is thought to affect approximately 85% of patients with CBSDH, and very high myopia (greater than 5 D) affects 50-76% of patients. Iridosymptomonas (trembling of the iris after eye movement) occurs in approximately 56% of patients, and spherophakia (a small, spherical lens prone to subluxation) occurs in 50% of patients (see Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [regular online] 2005; Mulvihill et al. J AAPOS 2001;5:311-315, each incorporated herein by reference in its entirety). Additional complications associated with CBSDH include cataract formation, chronic vitritis (inflammation of the vitreous humor) and chorioretinal inflammation, pupil closure due to acute and / or chronic angle-closure glaucoma, and (in children) amblyopia (lazy eye) (Sadiq et al., 2004, pp. 111-114, which is incorporated herein by reference in its entirety). et al.) Semin Ophthalmol 2013;28:313-320).

[0166] Evidence from a longitudinal retrospective study of 25 patients under the age of 24 with CBSDH suggests that lens dislocation can be prevented, or at least significantly reduced and delayed, in patients who consistently lower their tHcy levels from an early age (see Non-Patent Document 6, incorporated herein by reference in its entirety). Early Hcy-lowering treatment was also associated with a reduced risk of overall ocular complications, including worsening myopia. Supporting evidence comes from a case-control study of 32 CBSDH patients and 25 sibling controls, in which early Hcy-lowering treatment was associated with a significant reduction in ocular complications compared with patients treated later in life or who did not fully comply with treatment (see El Bashir et al. JIMD Rep 2015;21:89-95, incorporated herein by reference in its entirety).

[0167] The largest and longest longitudinal study to date of ocular outcomes in 25 patients with cobalamin C deficiency, also characterized by elevated tHcy levels, found that macular degeneration, optic nerve pallor, nystagmus, strabismus, and vascular changes were all present in the majority of patients.

[0168] Numerous studies in patients with CBSDH and the general population have linked elevated Hcy levels to myopia and lens dislocation (Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [regular online] 2005; Mudd et al. Am J Hum Genet 1985;37:1-31; Martinez-Gutierrez et al. Int Ophthalmol 2011;31:227-232; Suri et al. J Neurol Sci 2014;347:305-309; Mulvihill et al. J AAPOS, each of which is incorporated herein by reference in its entirety). 2001;5:311-315; Lieberman et al. Am J Ophthalmol 1966;61:252-255; Harrison et al. Ophthalmology 1998;105:1886-1890; Ramsey et al. Am J Ophthalmol 1972;74:377-385; Couser et al. Ophthalmic Genet 2017;38:91-94), iris tremens (see Mulvihill et al. J AAPOS 2001;5:311-315, which is incorporated herein by reference in its entirety), retinal arteriosclerosis (see Ghorbanihaghjo et al. Mol Vis, which is incorporated herein by reference in its entirety), and retinal vasculitis (see Ghorbanihaghjo et al. Mol Vis, which is incorporated herein by reference in its entirety). 2008;14:1692-1697), age-related macular degeneration (Javadzadeh et al., each of which is incorporated herein by reference in its entirety). al. Mol Vis 2010;16:2578-2584; Seddon et al. Am J Ophthalmol 2006;141:201-203; Coral et al. Eye(Lond) 2006;20:203-207; Axer-Siegel et al. Am J Ophthalmol 2004;137:84-89), age-related maculopathy (AMD) (see Heuberger et al. Am J Clin Nutr 2002;76:897-902; Huang et al. Sci Rep 2015;5:10585), cataracts (see Sen et al. al.) Indian J Clin Biochem 2008;23:255-257; Yousefi et al. Protein Pept Lett 2013;20:932-941), diabetic retinopathy (possibly secondary to renal disease) (Looker et al. Diabetologia 2003;46:766-772; Pusparajah et al. Front Physiol 2016;7:200), and patchy atrophy and optic nerve atrophy due to retinal vascular occlusion or non-arteritic ischemic optic neuropathy (Gerth et al. J AAPOS 2008;12:591-596; Stanger et al. Clin Chem Lab Med 2005;43:1020-1025; Cahill et al. Am J Ophthalmol 2003;136:1136-1150; Minniti et al. Eur J Ophthalmol 2014;24:735-743) (see Mudd et al. Am J Hum Genet 1985;37:1-31; Non-Patent Document 13; Mulvihill et al. J AAPOS 2001;5:311-315; Wright et al. Eye(Lond) 2008;22:989-993, each of which is incorporated herein by reference in its entirety).

[0169] A retrospective study of 629 patients with CBSDH found that lens dislocation usually occurred after age 2 years and was present by age 6 years in approximately 50% of untreated pyridoxine-nonresponsive patients and by age 10 years in 50% of untreated pyridoxine-responsive patients (see Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein in its entirety). The largest and longest longitudinal study to date of ocular outcomes in 25 patients with cobalamin C deficiency, also characterized by elevated tHcy levels, found that macular degeneration, optic nerve pallor, nystagmus, strabismus, and vascular changes were all present in the majority of patients (see Brooks et al., Ophthalmology. 2016 Mar;123(3):571-82, incorporated herein in its entirety).

[0170] Numerous studies in patients with CBSDH and the general population have linked elevated Hcy levels to myopia and lens dislocation (Yap S. Homocystinuria due to cystathionine beta-synthase deficiency. Orphanet Encyclopaedia [regular online] 2005; Mudd et al. Am J Hum Genet 1985;37:1-31; Martinez-Gutierrez et al. Int Ophthalmol 2011;31:227-232; Mulvihill et al. J AAPOS 2001;5:311-315; Sadiq et al. Semin Ophthalmol 2013;28:313-320; Lieberman et al. Am J Ophthalmol 1966;61:252-255; Harrison et al. Ophthalmology 1998;105:1886-1890; Ramsey et al. Am J Ophthalmol 1972;74:377-385; Couser et al. Ophthalmic Genet 2017;38:91-94), iris tremens (see Mulvihill et al., which are incorporated herein by reference in their entireties). et al. J AAPOS 2001;5:311-315), retinal arteriosclerosis (Ghorbanihaghjo et al. Mol Vis 2008;14:1692-1697, each of which is incorporated herein by reference in its entirety), age-related macular degeneration (Javadzadeh et al. Mol Vis 2010;16:2578-2584; Seddon et al. Am J Ophthalmol 2006;141:201-203; Coral et al. Eye(Lond) 2006;20:203-207; Axer-Siegel et al. Am J Ophthalmol 2004;137:84-89), age-related maculopathy (AMD) (see Heuberger et al. Am J Clin Nutr 2002;76:897-902; Huang et al. Sci Rep 2015;5:10585, both of which are incorporated herein in their entireties), cataracts (see Sen et al. Indian J Clin Biochem 2008;23:255-257; Yousefi et al. Protein Pept Lett 2013;20:932-941, both of which are incorporated herein in their entireties), diabetic retinopathy (possibly secondary to renal disease) (Looker et al. Diabetologia 2003;46:766-772; Pusparajah et al. Front Physiol 2016;7:200), and patchy atrophy and optic nerve atrophy due to retinal vascular occlusion or non-arteritic ischemic optic neuropathy (Gerth et al. J AAPOS 2008;12:591-596; Stanger et al. Clin Chem Lab Med 2005;43:1020-1025; Cahill et al. Am J Ophthalmol 2003;136:1136-1150; Minniti et al. Eur J Opt Neurol 2009;10:1162-1164, each of which is incorporated herein by reference in its entirety). Ophthalmol 2014;24:735-743) (see Mudd et al. Am J Hum Genet 1985;37:1-31; Non-Patent Document 13; Mulvihill et al. J AAPOS 2001;5:311-315; Wright et al. Eye(Lond) 2008;22:989-993, each of which is incorporated herein by reference in its entirety).

[0171] Studies investigating the association between Hcy levels and glaucoma have provided inconsistent results. Some studies have shown a positive relationship between Hcy levels and normal tension glaucoma, pseudoexfoliation glaucoma (PEXG), and primary open angle glaucoma (POAG), while others have not (Lieberman et al. Am J Ophthalmol 1966;61:252-255; Turkcu et al. Medicina (Kaunas) 2013;49:214-218; Vessani et al. Am J Ophthalmol 2003;136:41-46; Leibovitch et al. J Glaucoma 2003;12:36-39; Leibovitzh et al. Relationship between homocysteine ​​and intraocular pressure in men and women: a population-based study, each of which is incorporated herein by reference in its entirety). and women: A population-based study). Medicine (Baltimore) 2016;95:e4858; Micheal et al. (See et al.) Mol Vis 2009;15:2268-2278; Clement et al. J Glaucoma 2009;18:73-78; Cumulcu et al. BMC Ophthalmol 2006;6:6; Bleich et al. J Neural Transm (Vienna) 2002;109:1499-1504; Lee et al. Curr Eye Res 2017;1-6; Wang et al. Am J Ophthalmol 2004;137:401-406). However, loss of retinal ganglion cells (RGCs)—a common observation in individuals with glaucoma—was demonstrated in mice with endogenously elevated Hcy levels caused by CBS gene deletion, suggesting a likely link between glaucoma and elevated tHcy levels in CBSDH patients (see Ganapathy et al. Invest Ophthalmol Vis Sci 2009;50:4460-4470, incorporated herein by reference in its entirety).

[0172] 1. Mechanism Several mechanisms have been proposed to explain the effects of elevated Hcy levels on ocular health (see Non-Patent Document 13, which is incorporated herein by reference in its entirety). Mechanisms explaining the effects of elevated tHcy include vascular endothelial dysfunction, retinal ganglion cell apoptosis, extracellular matrix alterations, decreased lysyl oxidase activity and oxidative stress, as well as direct cytotoxic and pro-inflammatory effects of Hcy that appear to contribute to lens opacification and optic nerve damage.

[0173] Potential mechanisms include activation of N-methyl-D-aspartate (NMDA) receptors, leading to cellular calcium influx and increased reactive oxygen species (ROS) generation, both of which contribute to cataract formation. These changes, along with the direct cytotoxic effects of Hcy, can cause endothelial injury, which initiates thrombus formation and RGC apoptosis, leading to retinopathy and glaucoma. Elevated Hcy levels have also been shown to cause vasoconstriction and optic nerve atrophy by increasing asymmetric dimethylarginine (AMDA) levels, blocking nitric oxide synthase (NOS) activity and thereby reducing nitric oxide (NO) levels. Finally, accumulation of homocysteinylated proteins on the vascular wall can trigger anti-Hcy antibody production and an inflammatory response, leading to phagocytosis, oxidative stress, RGC apoptosis, and extracellular matrix (ECM) alterations. Collectively, these changes damage the vasculature, lens proteins, and optic nerve, ultimately causing visual dysfunction.

[0174] In patients with CBSDH, lens dislocation is thought to be primarily caused by degenerative changes in zonular fibers, particularly Cys-rich multidomain ECM proteins such as fibrillin-1 (see Sadiq et al. Semin Ophthalmol 2013;28:313-320; Hubmacher et al. Biochemistry 2011;50:5322-5332; Hubmacher et al. J Biol Chem 2005;280:34946-34955; Hubmacher et al. J Biol Chem 2010;285:1188-1198, each of which is incorporated herein by reference in its entirety).

[0175] In healthy individuals, the formation of numerous intradomain disulfide bonds within fibrillin-1 allows for the correct protein folding that is essential for structural integrity and function. Fibrillin-1 chains can then form intrachain disulfide bonds, leading to the assembly of high molecular weight multiprotein assemblies known as microfibrils (see Kinsey et al. J Cell Sci 2008;121:2696-2704; Hubmacher et al. Proc Natl Acad Sci USA 2008;105:6548-6553, both of which are incorporated herein by reference in their entireties).

[0176] This process is highly dependent on the interaction between fibrillin-1 and fibronectin (see Hubmacher et al. Biochemistry 2011;50:5322-5332, incorporated herein in its entirety). Microfibrils form a scaffold for the deposition of tropoelastin, an essential step in the formation of elastic fibers such as those found in skin, lung, blood vessels / arteries, ligaments, and the eye (see Hubmacher et al. J Biol Chem 2010;285:1188-1198, incorporated herein in its entirety). The importance of fibrillin-1 is exemplified by patients with Marfan syndrome—a condition caused by mutations in the fibrillin-1 gene—in which connective tissue dysfunction is associated with symptoms such as lens dislocation, organ prolapse, osteoporosis, and joint hypermobility (Suk et al. J Biol Chem 2004;279:51258-51265; Collod-Beroud et al. Hum, both of which are incorporated herein by reference in their entireties). See Mutat 2003;22:199-208).

[0177] In vitro studies have shown that the addition of Hcy to fibrillin-1 disrupts disulfide bond formation, which in turn leads to abnormal protein folding, increased susceptibility to proteolysis, and abnormal formation of ECM and elastic fibers (see Hubmacher et al. J Biol Chem 2010;285:1188-1198; Whiteman et al. Antioxid Redox Signal 2006;8:338-346, both of which are incorporated herein by reference in their entireties). The addition of Hcy to human skin fibroblasts has also been associated with a reduced form of fibronectin that bound suboptimally to fibrillin-1, thereby preventing microfibril formation (Hubmacher et al. Biochemistry 2011;50:5322-5332; Hubmacher et al. J Biol Chem 2011;50:5322-5332; Hubmacher et al. J Biol Chem 2011;50:5332-5332, both of which are incorporated herein by reference in their entireties). 2010;285:1188-1198).

[0178] In addition to lens dislocation, zonular fiber degeneration in CBSDH patients can lead to increased lens curvature, lens myopia, astigmatism, retinal detachment, strabismus, cataracts, and iris tremens (see Sadiq et al., Semin Ophthalmol 2013;28:313-320, incorporated herein in its entirety). If untreated, anterior lens dislocation can lead to acute pupil-closure glaucoma. In extreme cases, complete lens dislocation is associated with increased axial length, likely a compensatory response to blurred vision (see Mulvihill et al., J AAPOS 2001;5:311-315, incorporated herein in its entirety).

[0179] A retrospective study to examine the effect of Hcy-lowering therapy on clinical outcomes was conducted on 25 CBSDH cases detected in Ireland between 1971 and 1996, either by the National NBS Program or clinical presentation (see Non-Patent Document 6, incorporated herein by reference in its entirety). The majority of cases (24 / 25) were pyridoxine-unresponsive. As a result, treatment for most patients consisted of a Met-free Cys-supplemented diet and, if necessary, vitamin B 12 and folic acid supplementation. Treatment began before the patients were 6 weeks of age and compared with a different group in which treatment began at diagnosis and one control patient who never received treatment. The mean follow-up period was 14.3 years (range 2.5-23.4) for the group treated before 6 weeks of age and 14.7 years (range 11.7-18.8) for the other patients, for a total of 365.7 patient-treatment years. Of the 21 patients detected by NBS, 18 remained complication-free during treatment. Of these individuals, 15 / 18 had 20:20 vision, and 3 / 18 had increased myopia over the past 2 years.

[0180] Mudd et al. (incorporated herein in its entirety) Am. Consistent with the findings of [J Hum Genet 1985;37:1-31], lens dislocation in the late-diagnosed individuals occurred at approximately 2 years of age. Lens dislocation was not reported in any of the early-treated individuals who adhered well to treatment. Three of the "early-treated patients" (those with the highest levels of fHcy) had myopia worsening without lens dislocation, most likely due to the relatively high fHcy levels in this small patient group. This led the authors to suggest that progressive myopia may be the first sign of poor dietary compliance, before lens dislocation, despite the patients' claims to the contrary. The myopia worsening in these patients highlights how tenuous the balance is between neutral and negative clinical outcomes in these patients. All of the late-detected patients developed ectopia lentis, suggesting that treatment delays rather than prevents ectopia.

[0181] Lifetime median plasma fHcy levels were higher in myopic patients than in non-myopic patients (18, 18, and 48 μmol / L vs. 11 μmol / L, respectively). Of the three patients identified by NBS who developed complications in the group in which treatment began at diagnosis, all were non-compliant with the diet. Overall, 6 / 24 patients had lens dislocation; of these, 2 had early diagnosis but were non-compliant with the diet, 4 had late diagnosis, and 1 was never treated. Consistent with the findings of Mudd et al. (See Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety), lens dislocation in late-diagnosed individuals (i.e., patients presenting with complications after 2 years of age) occurred at approximately 2 years of age. When the study was published, lens dislocation was not reported by any of the early-treated individuals who were well-compliant with treatment.

[0182] Adherent patients maintained fHcy levels well below 120 μmol / L of tHcy equivalent. However, all patients were under 24 years of age at the time of publication, and many were still pediatric patients. Adherence to a protein-restricted diet has been shown to decline rapidly from adolescence to adulthood. This delicate balance suggests that the modest reductions in tHcy levels achieved by these patients may delay, rather than prevent, the onset of symptoms as they age.

[0183] These results (see Non-Patent Document 6; Mudd et al. Am J Hum Genet 1985;37:1-31, each incorporated herein in its entirety) were supported by results from a similar case-control study conducted in Qatar (see El Bashir et al. JIMD Rep 2015;21:89-95, incorporated herein in its entirety), which reported on outcomes, including visual impairment, in 32 CBSDH cases and 25 sibling controls. The mean age of subjects was 11.2 years (range 0.6-29), and 56% were male. Overall, 9 / 32 cases (28%) were diagnosed by NBS and treated at 1 month of age. The remainder were diagnosed between 14 and 240 months of age. tHcy and Met levels were significantly lower in those diagnosed through NBS compared with those diagnosed clinically. This was likely due to good dietary and medication adherence early in life. None of the nine cases identified by NBS had vision problems at the time the study was published, compared with 18 (78%) in the later-diagnosed group (p<0.001 between groups). However, as with the Irish study of 25 patients mentioned above, the patients in this study ranged in age from 0.6 to 29 years, and long-term complications remain unknown.

[0184] Comparison of the data of Yap and Naughten (see Non-Patent Document 6, incorporated herein by reference in its entirety) with the Kaplan-Meier curves generated by Mudd et al. showed that the proportion of compliant "early treated" patients with lens dislocation and osteoporosis was significantly lower than that expected for untreated CBSDH patients (p≦0.001).

[0185] Therefore, elevated Hcy levels are considered to be a strong independent risk factor for ocular complications, particularly lens dislocation, in patients with CBSDH and in the general population (e.g., Yap et al. and Mudd et al.), emphasizing the need for early CBSDH diagnosis and treatment, as well as patient adherence to treatment.

[0186] B. Skeletal complications CBSDH is associated with an increased risk of osteoporotic fractures, which may be due in part to low bone mineral density (Mudd et al., 2004; Mudd et al., 2005; Mudd et al., 2006; Mudd et al., 2007; Mudd et al., 2009; Mudd et al., 2010; Mudd et al., 201 ... al. and Weber et al. Mol Genet Metab 2016;117:351-354).

[0187] A retrospective chart review of data from 19 patients with CBSDH over an 8-year period found that low bone mineral density (BMD) was common among both pediatric and adult CBSDH patients (see Weber et al.). This study suggested that the natural increase in bone mass during childhood and adolescence, a crucial period for skeletal growth, is defective in CBSDH and may negatively affect the achievement of peak bone mass. This study also highlighted that even well-adhered patients with moderately elevated tHcy levels, only 5 times higher than the normal range, already suffer from poor skeletal clinical outcomes in childhood.

[0188] According to Mudd et al., 80% of patients with CBSDH develop osteoporosis before the age of 30. Furthermore, elevated Hcy levels are associated with an increased risk of osteoporotic fractures, even in patients without CBSDH (Sato et al. Bone 2005;36:721-726; van Meurs et al. N Engl J Med 2004;350:2033-2041; McLean et al., each of which is incorporated herein by reference in its entirety). et al.) N Engl J Med 2004;350:2042-2049).

[0189] A retrospective medical record review of data from 19 subjects (9 men, ages 3.5-49.2 years) who underwent clinical DXA bone mineral density measurements between 2002 and 2010 found that low BMD was common among both pediatric and adult CBSDH patients (see Weber et al. Mol Genet Metab 2016;117:351-354, incorporated herein by reference in their entirety). At the time of the initial DXA scan, the mean lumbar spine (LS) BMD Z-score was -1.2 ± 1.3, and the hip BMD Z-score was -0.89 ± 0.4; both were significantly lower than 0 (the expected mean Z-score for the general population) with p = 0.002 and 0.02, respectively. At the time of diagnosis, the LS BMD Z-score was -1.26 ± 1.4 for patients under 21 years of age and -1.06 ± 1.1 for adults. Overall, 38% of patients had low BMD for their age (defined by a Z-score of -2 or less). Both tHcy and Met levels were positively associated with LS BMD Z-score in multiple linear regression models (see Weber et al., Mol Genet Metab 2016;117:351-354, incorporated herein by reference in its entirety). The mean tHcy level for these 19 individuals was only 59.2 μmol / L, and the majority of the 19 patients were children. This study suggests that the natural accrual of bone mass during childhood and adolescence, a critical period for skeletal growth, is defective in CBSDH and may negatively affect the achievement of peak bone mass. This study also highlights that even well-adhered patients with moderately elevated tHcy levels, only five times higher than the normal range, already suffer from poor skeletal clinical outcomes in childhood.

[0190] Previous studies have demonstrated a clear relationship between Hcy levels and fracture risk in elderly populations (see Sato et al., Bone 2005;36:721-726; van Meurs et al., N Engl J Med 2004;350:2033-2041; McLean et al., N Engl J Med 2004;350:2042-2049, each of which is incorporated herein by reference in its entirety). Results from two prospective population-based studies involving 2,406 subjects aged 55 years or older showed that the age- and sex-adjusted risk of fracture increased by 30% for each 1 SD increase in tHcy levels (see van Meurs et al., N Engl J Med 2004;350:2033-2041, incorporated herein by reference in its entirety). Homocysteine ​​levels in the highest age-specific quartile were associated with a 1.9-fold increase in fracture risk. The association between homocysteine ​​levels and fracture risk appeared to be independent of bone mineral density and other potential risk factors for fracture. Elevated homocysteine ​​levels were a strong independent risk factor for osteoporotic fractures in elderly men and women from the general population, similar in magnitude to established risk factors for fracture and cardiovascular disease (see van Meurs et al. N Engl J Med 2004;350:2033-2041, incorporated herein by reference in its entirety). Furthermore, a US prospective study of 825 men and 1174 women (HOPE-2 trial substudy) found that serum tHcy levels in the highest quartile were associated with a 1.9-fold increased risk of hip fracture among women and a 4-fold increased risk among men compared with serum tHcy levels in the lowest quartile (Sawka et al., 2014, incorporated herein by reference in its entirety). (See van Meurs et al., N Engl J Med 2004;350:2033-2041; McLean et al., N Engl J Med 2004;350:2042-2049; Sawka et al., Arch Intern Med. 2007 Oct 22;167(19):2136-9, each of which is incorporated herein by reference in its entirety.) The association between tHcy levels and fracture risk was independent of BMD and other potential risk factors for fracture.

[0191] Consistent with these results, a study of 433 stroke patients over 65 years of age found that the age-adjusted incidence rate per 1,000 person-years for femoral neck fractures increased approximately linearly from 2.89 in the lowest quartile of Hcy levels to 27.87 in the highest quartile (see Sato et al., Bone 2005;36:721-726, incorporated herein by reference in its entirety). Collectively, these results suggest that elevated Hcy levels are a strong independent risk factor for osteoporotic fractures in elderly men and women.

[0192] Skeletal abnormalities are not present at birth and are uncommon in infants and very young children (see Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein by reference in their entireties). The first signs of skeletal involvement are usually genu valgum and pes cavus, often accompanied by elongation of the long bones, a typical feature of Marfan syndrome, with onset near puberty (see Non-Patent Document 12, incorporated herein by reference in their entireties). Osteoporosis, particularly of the vertebrae and long bones, is common in patients with CBSDH and can lead to scoliosis / kyphosis and / or vertebral collapse (see Mudd et al., Am J Hum Genet 1985;37:1-31; Weber et al., Mol Genet Metab 2016;117:351-354, incorporated herein by reference in their entireties). Other skeletal manifestations may include Marfanoid physiognomy caused by prominent upper teeth and a high palate, as well as anterior chest wall deformities such as pectus excavatum or pectus carinatum (see Non-Patent Document 12; Sweetser et al., N Engl J Med 2016;375:1879-1890; Brenton et al., J Bone Joint Surg Br 1972;54:277-298, each of which is incorporated herein by reference in its entirety). Because of these shared skeletal features between Marfan syndrome and CBSDH, patients with CBSDH are sometimes mischaracterized as Marfan patients.

[0193] A study of 25 Irish CBSDH patients followed for 25 years found that the risk of osteoporosis was significantly lower in patients identified through newborn screening who were well-adherent to Hcy-lowering treatment (diet, vitamins, and / or betaine) compared with non-adherent or late-diagnosed patients (see Non-Patent Document 6). Supporting evidence for these results comes from a small Korean study of five CBSDH patients with excellent long-term metabolic control. In this study, patients who received early Hcy-lowering therapy had fewer skeletal abnormalities than patients diagnosed later (see Lim et al., Osteoporos Int 2013, 24:2535-2538, incorporated herein by reference in its entirety). Finally, in a mouse model of CBSDH, normalization of tHcy levels by treatment with CBS ET was associated with the prevention of osteoporosis (see Majtan et al., "Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria," FASEB J 2017, incorporated herein by reference in its entirety).

[0194] The exact mechanisms leading to low BMD and skeletal fragility in CBSDH patients are not fully understood (Weber et al., 2004, pp. 111-114, both of which are incorporated herein by reference in their entireties). al.) Mol Genet Metab 2016;117:351-354; Rim Lim JS, Lee DH. Changes in bone mineral density and body composition of children with well-controlled homocystinuria caused by CBS deficiency. deficiency). Osteoporos Int 2013;24:2535-2538). However, many of the connective tissue disorders in CBSDH patients resemble those seen in Marfan syndrome, a connective tissue disorder caused by mutations in the fibrillin-1 gene and characterized by features including elongation of long bones and osteoporotic fractures (see Hubmacher et al. Biochemistry 2011;50:5322-5332; Hubmacher et al. J Biol Chem 2010;285:1188-1198, both of which are incorporated herein by reference in their entireties). Elevated Hcy levels are thought to lead to bone fragility and fractures through two distinct pathways (see Non-Patent Document 15, which is incorporated herein by reference in its entirety). The first leads to reduced natural bone accrual during childhood and adolescence via impaired fibrillin assembly. The second pathway leads to impaired bone remodeling, resulting in brittle bones through reduced collagen cross-linking (see, e.g., J. Clin. Invest. 1973;52:2571-2578, both of which are incorporated herein by reference in their entireties). Collectively, these data suggest that the natural accrual of bone mass during childhood and adolescence, a critical period for skeletal growth, is defective in patients with CBSDH, and that this negatively impacts the achievement of peak bone mass.

[0195] Furthermore, in elderly populations, a strong relationship exists between Hcy levels and fracture risk (see Sato et al., Bone 2005;36:721-726; van Meurs et al., N Engl J Med 2004;350:2033-2041; McLean et al., N Engl J Med 2004;350:2042-2049, each of which is incorporated herein by reference in its entirety). Results from two international prospective population-based studies involving 2,406 subjects aged 55 years or older showed that homocysteine ​​levels in the highest age-specific quartile were associated with a 1.9-fold increase in fracture risk (see van Meurs et al., N Engl J Med 2004;350:2033-2041, incorporated herein by reference in its entirety). Elevated homocysteine ​​levels were a strong independent risk factor for osteoporotic fractures in elderly men and women from the general population, similar in magnitude to established risk factors for fractures (low bone mineral density, cognitive impairment, recent falls) and cardiovascular disease (see van Meurs et al., N Engl J Med 2004;350:2033-2041, incorporated herein by reference in its entirety). Furthermore, a prospective US study of 1,999 subjects (the HOPE-2 trial substudy) found that serum tHcy levels in the highest quartile were associated with a 1.9-fold increased risk of hip fracture among women and a 4-fold increased risk among men compared with serum tHcy levels in the lowest quartile (see Sawka et al., Arch Intern Med. 2007 Oct. 22;167(19):2136-9, incorporated herein by reference in its entirety). The association between tHcy levels and fracture risk was independent of BMD and other potential risk factors for fracture (van Meurs et al. N Engl J Med 2004;350:2033-2041; McLean et al. N Engl J Med 2004;350:2042-2049; Sawka et al. Arch, each of which is incorporated herein by reference in its entirety). Intern Med. 2007 Oct. 22;167(19):2136-9). Consistent with these results, a study of 433 stroke patients over 65 years of age found that the age-adjusted incidence rate per 1,000 person-years for femoral neck fractures increased approximately linearly from 2.89 in the lowest quartile of Hcy levels to 27.87 in the highest quartile (see Sato et al., Bone 2005;36:721-726, incorporated herein by reference in its entirety). Collectively, these results suggest that elevated Hcy levels are a strong independent risk factor for osteoporotic fractures in elderly men and women.

[0196] The exact mechanisms leading to low BMD and skeletal fragility in CBSDH patients are not fully understood (Weber et al., 2004, pp. 111-114, both of which are incorporated herein by reference in their entireties). al.) Mol Genet Metab 2016;117:351-354; Rim Lim JS, Lee DH. Changes in bone mineral density and body composition of children with well-controlled homocystinuria caused by CBS deficiency. deficiency). Osteoporos Int 2013;24:2535-2538). However, many of the connective tissue disorders in CBSDH patients resemble those seen in Marfan syndrome, a connective tissue disorder caused by mutations in the fibrillin-1 gene and characterized by features including elongation of long bones and osteoporotic fractures (see Brenton et al. J Bone Joint Surg Br 1972;54:277-298; Hubmacher et al. Biochemistry 2011;50:5322-5332; Hubmacher et al. J Biol Chem 2010;285:1188-1198, each of which is incorporated herein by reference in its entirety).

[0197] In healthy individuals, fibrillin-1, together with collagen and elastin polymers, assembles to form the ECM, the architectural scaffold for bone formation, homeostasis, and repair (see Olivieri et al., Fibrogenesis Tissue Repair 2010;3:24, incorporated herein by reference in its entirety). Studies have shown that elevated tHcy levels can lead to structural modifications of fibrillin-1 fragments, preventing multimerization and leading to fibrillin-1 degradation (see Hubmacher et al., J Biol Chem 2005;280:34946-34955; Hubmacher et al., J Biol Chem 2010;285:1188-1198, both incorporated herein by reference in their entirety). This process is further impaired by homocysteinylation of fibronectin, which prevents the formation of fibronectin-fibrillin complexes required for fibrillin-1 multimerization (see Hubmacher et al., Biochemistry 2011;50:5322-5332, incorporated herein by reference in its entirety). These findings suggest that elevated Hcy levels have a detrimental effect on ECM formation.

[0198] In healthy individuals, fibrillin aggregates (i.e., microfibrils) play an important role in bone mineralization through the storage and activation of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs) (see Nistala et al., Ann NY Acad Sci 2010;1192:253-256; Nistala et al., J Biol Chem 2010;285:34126-34133, both of which are incorporated herein by reference in their entireties). Impaired activation of TGF-beta and BMPs could potentially contribute to the skeletal phenotypes observed in both Marfan syndrome and CBSDH and may also reduce bone mineral density, as observed in milder forms of CBSDH (see Herrmann et al., Clin Chem 2005;51:2348-2353, both of which are incorporated herein by reference in their entireties). Moreover, there is in vivo and in vitro evidence that Hcy may weaken bone strength through reduced collagen cross-linking (see Kang et al. J Clin Invest 1973;52:2571-2578, incorporated herein by reference in its entirety). Collectively, these data suggest that the natural accrual of bone mass during childhood and adolescence, a critical period for skeletal growth, is defective in patients with CBSDH, and that this may negatively affect the achievement of peak bone mass.

[0199] In addition to its effects on bone deposition, elevated Hcy levels increase the rate of bone remodeling by increasing osteoclast (OC) activity and decreasing osteoblast (OB) activity (see Non-Patent Document 15; Herrmann et al., Clin Chem 2005; 51:2348-2353; Vacek et al., Clin Chem Lab Med 2013; 51:579-590; Vijayan et al., J Endocrinol 2017; 233:243-255, each of which is incorporated herein by reference in its entirety). An imbalance between OB and OC activity can lead to brittle bones and increased fracture incidence. The mechanisms leading to Hcy-mediated decline in OB activity are believed to include decreased bone blood flow (as a result of decreased NO availability) (see Tyagi et al. Vasc Health Risk Manag 2011;7:31-35, incorporated herein in its entirety) and increased OB apoptosis rates (Figure 2 and Table 14) (see Kim et al. Bone 2006;39:582-590, both incorporated herein in their entirety). The mechanism leading to enhanced OC activity is thought to be elevated levels of intracellular ROS, which enhance both OC differentiation and OC activity via increased matrix metalloproteinase (MMP) activity (see Vacek et al. Vasc Health Risk Manag 2011;7:31-35, incorporated herein in its entirety). al. Clin Chem Lab Med 2013;51:579-590) and inhibition of OC apoptosis (see Non-Patent Document 15; Herrmann et al. Clin Chem 2005;51:2348-2353; Koh et al. J Bone Miner 2013;51:579-590, each of which is incorporated herein by reference in its entirety). Res 2006;21:1003-1011). Indeed, a recent study in CD1 mice fed a high-Hcy diet showed that short-term (7 days) Hcy administration was associated with loss of tissue mineral density (TMD) and increased OC numbers, whereas long-term Hcy administration (30 days) led to OC reprogramming, apoptosis, and calcification, restoring TMD but impairing tissue biomechanical properties (see Vijayan et al. J Endocrinol 2017;233:243-255, incorporated herein by reference in its entirety).

[0200] Thus, elevated Hcy levels can lead to bone fragility and fractures through two distinct pathways (see Non-Patent Document 15, which is incorporated herein by reference in its entirety). The first pathway leads to reduced natural bone mass accrual during childhood and adolescence through impaired ECM formation and suppressed fibrillin-1-associated TGF-beta and BMP activation. The second pathway leads to impaired bone remodeling through increased OC activity and decreased OB activity, resulting in fragile bones.

[0201] Elevated Hcy levels are associated with increased oxidative stress in the bone microenvironment. Increased ROS induce osteoblast apoptosis, thereby reducing osteoblast development. This increased oxidative stress further reduces NO availability through the generation of superoxide anions, which can also reduce bone blood flow and angiogenesis. The ROS generated by this process activate osteoclastogenesis through monocyte fusion, further contributing to the loss of BMD and leading to osteoporosis.

[0202] A recent study in neonatal CBS knockout (KO) mice maintained on a standard rodent chow diet without Met restriction found that subcutaneous administration of CBS ET using recombinant PEGylated human truncated CBS (PEG-CBS) for 5 months prevented bone mineral density loss in these animals and could normalize these values ​​in animals treated later in life (see Majtan et al., "Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria." FASEB J 2017, incorporated herein by reference in its entirety). In this study, the body composition changes characteristic of both the KO model and CBSDH patients were prevented. tHcy and Cys levels were normalized, Cth levels were elevated, and the SAM / SAH ratio improved in both plasma and tissues.

[0203] Supporting evidence for the effect of Hcy reduction on skeletal outcomes comes from a 25-year study of 25 Irish patients with CBSDH (see Non-Patent Document 6, incorporated herein by reference in its entirety). In this study, osteoporosis (diagnosed by radiology, not DXA) was present in one of three treatment-noncompliant patients identified by NBS and in one of four patients diagnosed late (at age 2 years). None of the 18 patients who adhered to early treatment (from age 6 weeks) showed signs of osteoporosis.

[0204] A small study of five CBSDH patients (three boys and three girls), all diagnosed at a young age (three at NBS and two at age 7 years), with good metabolic control over a 3.4-year period, was performed in Korea (see Lim et al., Osteoporos Int 2013;24:2535-2538, incorporated herein by reference in its entirety). The mean plasma tHcy level at diagnosis was 34.3 ± 52.6 (13-78.6) μmol / L. Plasma Met was 716 ± 1347.6 (24.3-1566) μmol / L, and treatment consisted of a low-Met diet supplemented with pyridoxine, betaine, and folic acid. Body composition measurements and BMD for all patients were within the normal range for the Korean population, and no significant changes in skeletal morphology were observed over time. Three patients (60%) had mild scoliosis of the TL vertebra (Cobb angles 7.3°, 7.6°, and 10.3°), and three patients reported four fractures. Of these, two were caused by sports injuries and one by a traffic accident. Two cases of mild compression fractures of the lumbar vertebrae were detected by radiography, and a history of severe back pain was recorded. Patients diagnosed early showed fewer skeletal abnormalities than patients diagnosed later. However, this study showed that even patients diagnosed early by NBS, who adhered to dietary treatment and had only slightly to moderately elevated tHcy levels, already displayed skeletal abnormalities and multiple fractures as children.

[0205] Taken together, these findings suggest a beneficial effect of early Hcy-lowering treatment on skeletal outcomes in patients with CBSDH. In compliant patients, tHcy levels decreased but did not normalize, and fewer skeletal abnormalities were present in these patients; however, it should be noted that significant negative clinical outcomes (osteoporosis and fractures) were observed in this much younger patient population.

[0206] C. Vascular complications The relationship between CBSDH and vascular disease was first demonstrated in 1985 in an epidemiological study of patients with moderate to severely elevated Hcy levels due to homozygous CBSDH (see Mudd et al. Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety).

[0207] Thromboembolism is a leading cause of morbidity and premature mortality in patients with CBSDH (see Mudd et al. Am J Hum Genet 1985;37:1-31; Karaca et al. Gene 2014;534:197-203; Yap S. J Inherit Metab Dis 2003;26:259-265, each of which is incorporated herein by reference in its entirety). The overall rate of thromboembolic events in patients with untreated CBSDH is approximately 10% per year (see Cattaneo M. Semin Thromb Hemost 2006;32:716-723, entirely incorporated herein by reference), with the risk increasing after surgery and during or immediately after pregnancy (see Mudd et al. Am J Hum Genet 1985;37:1-31; Novy et al. Thromb Haemost 2010;103:871-873, both entirely incorporated herein by reference). Although thromboembolism can affect any blood vessel, venous thrombosis (particularly CSVT) is more common than arterial thrombosis in patients with CBSDH (see Mudd et al. Am J Hum Genet 1985;37:1-31; Karaca et al. Gene 2014;534:197-203; Eslamiyeh et al. Iran J Child Neurol 2015;9:53-57; Saboul et al. J Child Neurol 2015;30:107-112, each of which is incorporated herein by reference in its entirety). Cerebrovascular accidents, particularly CSVT, have been described in infants (see Mahale et al. J Pediatr Neurosci. 2017 Apr-June;12(2):206-207, incorporated herein in its entirety), but more typically present in young adults (see Yap et al. Arterioscler Thromb Vasc Biol 2001;21:2080-2085, incorporated herein in its entirety).

[0208] The risk of a thromboembolic event was approximately 25% by age 16 and 50% by age 29. In 1999, Hankey et al. 12 reported that the deficiency of α-glucan (α-glucan deficiency) is associated with a high risk of premature cardiovascular (CV) disease, affecting half of all homozygotes by age 30 (see Hankey et al., Lancet 1999;354:407-413, incorporated herein in its entirety). The only biochemical alteration common to all three disorders is elevated serum Hcy levels (often greater than 100 μmol / L) (see Faeh et al., Swiss Med Wkly 2006;136:745-756, incorporated herein in its entirety). Several reports have described how treatments that lower tHcy levels significantly reduce the incidence of vascular events, a major cause of morbidity in patients with CBSDH (see Yap et al. J Inherit Metab Dis 2001;24:437-447; Wilcken DE, Wilcken B. The natural history of vascular disease in homocystinuria and the effects of treatment. J Inherit Metab Dis 1997;20:295-300, both of which are incorporated herein by reference in their entireties). Since then, several other studies have demonstrated an increased risk of vascular events, particularly venous thrombosis, in patients with CBSDH (see Karaca et al. Gene 2014;534:197-203; Kelly et al. Neurology 2003;60:275-279; Lussana et al. Thromb Res 2013;132:681-684; Magner et al. J Inherit Metab Dis 2011;34:33-37, each of which is incorporated herein by reference in its entirety).

[0209] Elevated plasma tHcy levels are a strong predictor of vascular disease risk factors and mortality in patients with coronary artery disease both with and without CBSDH (Mudd et al. Am J Hum Genet 1985;37:1-31; Karaca et al. Gene 2014;534:197-203; Kelly et al. Neurology 2003;60:275-279; Faeh et al. Swiss Med Wkly 2006;136:745-756; Boushey et al. JAMA 1995;274:1049-1057; Clarke R et al. JAMA 2009;136:745-756; Boushey et al. JAMA 2009;136:745-756; Clarke R et al. JAMA 2009;136:745-756; each of which is incorporated herein by reference in its entirety). 2002;288:2015-2022; Hankey et al. Lancet 1999;354:407-413; Khan et al. Stroke 2008;39:2943-2949; Graham et al. The European Concerted Action Project. JAMA 1997;277:1775-1781; Clarke et al. N Engl J Med 1991;324:1149-1155; Clarke et al. Ir J Med Sci 1992;161:61-65; Woodward et al. Blood Coagul Fibrinolysis 2006;17:1-5; Refsum et al. al.) Annu Rev Med 1998;49:31-62; Yoo et al. Stroke 1998;29:2478-2483; Selhub et al. N Engl J Med 1995;332:286-291; Wald et al. BMJ 2002;325:1202; Bautista et al. J Clin Epidemiol 2002;55:882-887; Brattstrom et al. (See McCully KS.) Atherosclerosis 1990;81:51-60; Lussana et al. Thromb Res 2013;132:681-684; Casas et al. Lancet 2005;365:224-232; McCully KS. Am J Pathol 1969;56:111-128; Magner et al. J Inherit Metab Dis 2011;34:33-37; Wilcken et al. J Clin Invest 1976;57:1079-1082; Nygard et al. N Engl J Med 1997;337:230-236). Although there is evidence for a relationship between tHcy levels and CV risk (see Boushey et al. JAMA 1995;274:1049-1057, which are incorporated herein by reference in their entireties), the relationship between tHcy and stroke / peripheral arterial disease is considerably stronger (see Clarke et al. JAMA 2002;288:2015-2022; Khan et al. Stroke 2008;39:2943-2949; Wald et al. BMJ 2002;325:1202; Casas et al. Lancet 2005;365:224-232; Brattstrom et al. Haemostasis 1989;19 (See Supplementary Note 1:35-44.) Large studies in the general population (NORVIT, HOPE-2, VITATOPS) initially concluded that lowering Hcy levels had only a modest effect on major vascular events and recurrent cardiovascular disease, but further, more specific analyses of the data clearly demonstrated the clinical benefit of lowering tHcy on stroke.

[0210] There is considerable evidence that lowering Hcy reduces stroke risk in the general population with mildly elevated tHcy levels (see Saposnik et al. Stroke 2009;40:1365-1372; Huo et al. JAMA 2015;313:1325-1335; Lonn et al. N Engl J Med 2006;354:1567-1577; Hankey et al. Lancet Neurol 2012;11:512-520; Spence JD, Lancet Neurol. 2007 Sep;6(9):830-8, each of which is incorporated herein by reference in its entirety). The 5552-patient HOPE-2 study (see Saposnik et al. Stroke 2009;40:1365-1372, incorporated herein in its entirety) showed that a small reduction in tHcy levels (3 mmol / L vs. placebo) led to a significant reduction in stroke incidence (27% relative risk reduction, 1.3% absolute risk reduction), suggesting that even small reductions in tHcy levels can be beneficial. This effect was most pronounced in patients with baseline Hcy in the upper quartile, with an absolute risk reduction of 4.3%. Whether Hcy reduction affects overall CV outcomes in patients with mildly elevated tHcy and no CBSDH remains unclear (see Marti-Carvajal et al. Stroke 2009;40:1365-1372, incorporated herein in its entirety). al. Cochrane Database Syst Rev 2015;1:CD006612), the vascular benefit has been consistently demonstrated in patients with CBSDH (see Non-Patent Document 6; Eslamiyeh et al., each of which is incorporated herein by reference in its entirety). et al.) Iran J Child Neurol 2015;9:53-57; Saboul et al. J Child Neurol 2015;30:107-112; Yap et al. Arterioscler See Thromb Vasc Biol 2001;21:2080-2085; Woods et al. BMJ Case Rep 2017; Wilcken et al. J Inherit Metab Dis 1997;20:295-300; Yap et al. Semin Thromb Hemost 2000;26:335-340; Ruhoy et al. Pediatr Neurol 2014;50:108-111).

[0211] 1. Mechanism Several studies have shown that elevated Hcy levels contribute to the development of atherosclerosis or thrombosis through oxidative stress-related mechanisms, including NF-κB-mediated inflammation and immune activation (see Rodriguez-Ayala et al. Atherosclerosis 2005;180:333-340; van Guldener et al. Curr Hypertens Rep 2003;5:26-31, both of which are incorporated herein by reference in their entireties) (Faverzani et al. Cell Mol Neurobiol 2017; Nowak et al. Arterioscler Thromb Vasc Biol 2017;37:e41-e52; Vanzin et al. Mol Genet Metab, each of which is incorporated herein by reference in its entirety). 2011;104:112-117; Vanzin et al. Gene 2014;539:270-274; Vanzin et al. Cell Mol Neurobiol 2015;35:899-911). Medical insults leading to thrombosis include Hcy-mediated endothelial dysfunction (see Jiang et al. Arterioscler Thromb Vasc Biol 2005;25:2515-2521; Hossain et al. J Biol Chem 2003;278:30317-30327; Cai et al. Blood 2000;96:2140-2148; Zhang et al. J Biol Chem 2001;276:35867-35874; Papapetropoulos et al. Proc Natl Acad Sci USA 2009;106:21972-21977; Szabo et al. al.) Br J Pharmacol 2011;164:853-865; Chiku et al. J Biol Chem 2009;284:11601-11612; Wang et al. Antioxid Redox Signal 2010;12:1065-1077; Saha et al. FASEB J 2016;30:441-456; Ebbing et al. JAMA 2008;300:795-804; Bonaa et al. N Engl J Med 2006;354:1578-1588; Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612; Celermajer et al. J Am Coll Cardiol 1993;22:854-858; Rubba et al. Metabolism 1990;39:1191-1195), enhanced coagulation pathways (Spence JD. Int J Stroke. 2016 Oct;11(7):744-7; Fryer et al., each of which is incorporated herein by reference in its entirety). (See, e.g., Kelly et al., Arterioscler Thromb 1993;13:1327-1333; Lentz et al., J Clin Invest 1991;88:1906-1914) and is thought to be caused by increased vasodilation. Such prothrombotic mechanisms are similar to those observed in Marfan patients (Kelly et al., Arterioscler Thromb 1993;13:1327-1333; Lentz et al., J Clin Invest 1991;88:1906-1914), each of which is incorporated herein by reference in its entirety. (See also: Tripathi P. International Cardiovascular Forum J 2016;6:13; van Guldener et al. Curr Hypertens Rep 2003;5:26-31; Hackam et al. JAMA 2003;290:932-940; Baumbach et al. Circ Res 2002;91:931-937; Evangelisti et al. Int J Cardiol 2009;134:251-254; de Valk et al. Stroke 1996;27:1134-1136).

[0212] A causal relationship between tHcy levels and CV risk derived from a meta-analysis of data from 27 studies (over 4000 patients) showed a graded risk of CV, cerebrovascular, and peripheral atherosclerosis, with a 5 μM increase in Hcy conferring an 80% increased risk in women and a 60% increased risk in men (see Boushey et al. JAMA 1995;274:1049-1057, incorporated herein by reference in its entirety). A meta-analysis of data from 30 prospective or retrospective studies involving 5,073 ischemic heart disease (IHD) events and 1,113 strokes found that Hcy levels 25% lower than normal (approximately 3 μmol / L) (corrected for regression dilution bias) were associated with an 11% (odds ratio (OR), 0.89; 95% CI, 0.83-0.96) lower IHD risk and a 19% (OR, 0.81; 95% CI, 0.69-0.95) lower stroke risk (see Clarke R, et al. JAMA 2002;288:2015-2022, incorporated herein by reference in its entirety).

[0213] Studies in patients with and without pre-existing vascular disease demonstrated elevations in plasma Hcy (above the highest values ​​in comparable healthy controls) after Met challenge in 1 / 21 subjects with MI (5%), 14 / 37 subjects with aortoiliac artery disease (38%), and 17 / 53 subjects with cerebrovascular disease (32%). This suggests that the association between Hcy levels and peripheral arterial disease (PAD) and stroke is significantly greater than the association between Hcy levels and MI (Brattstrom et al., Haemostasis 1989;19 Suppl 1:35-44). An independent grading association between Hcy levels and stroke was described in a prospective study conducted among a UK cohort of 457 stroke patients and 179 control subjects from the same community (Khan et al., Stroke 2008;39:2943-2949). The highest Hcy levels were observed in patients with small vessel disease (SVD) (16.2 μmol / L vs. 11.8 μmol / L in stroke-free control subjects, p<0.001, after adjusting for age, sex, vascular risk factors, vitamin levels, and renal function). Within SVD cases, the highest Hcy levels were observed in individuals with lacunar infarcts and confluent white matter lesions. Furthermore, there was a correlation between Hcy levels and the severity of white matter lesions (r=-0.225; p<0.001).

[0214] These findings were further supported by Mendelian randomization studies demonstrating a genetic association between MTHFR polymorphisms regulating Hcy metabolism and stroke risk (see Casas et al., Lancet 2005;365:224-232, incorporated herein by reference in its entirety). A literature search for all relevant studies on the association between Hcy levels and MTHFR TT and CC polymorphisms on stroke risk identified 111 studies involving 15,635 individuals without cardiovascular disease (CVD). The weighted mean difference in Hcy levels between TT and CC homozygotes was 1.93 μmol / L (95% CI 1.38-2.47). Based on results from a previous meta-analysis of prospective studies in which a 5 μmol / L increase in plasma Hcy levels corresponded to an OR of stroke of 1.59 (1.29 to 1.96), a 1.93 μmol / L increase in Hcy levels in healthy individuals with the TT genotype would result in a predicted OR of stroke of 1.20 (1.10 to 1.31) (Wald et al., BMJ, 2014, incorporated herein by reference in its entirety). 2002;325:1202). Consistent with this result, Khan et al. al.) reported an OR of 1.26 (1.14-1.40) for stroke in TT homozygotes compared with CC homozygotes, regardless of age group, ethnicity, or geographic location (p=0.29). Collectively, these results suggested a causal role for elevated Hcy levels in stroke incidence in the general population.

[0215] Because vascular disease has been investigated in much smaller studies than those performed in the general population, the moderate elevations in serum Hcy are typical for patients with heterozygous CBSDH. According to Mudd et al., the risk of vascular events in patients with mildly elevated tHcy levels (less than 5% by age 50 years) due to heterozygous CBSDH is similar to that in the general population (Mudd et al., Am J Cardiol 1999, 144:111-112, which is incorporated herein by reference in its entirety). J Hum Genet 1981;33:883-893. Consistently, ultrasound studies in individuals with homozygous and heterozygous CBSDH have found impaired endothelial function in systemic arteries in homozygous children as young as 4 years of age, whereas endothelial function was largely unaffected in heterozygous adults (Celermajer et al. J Am Coll Cardiol 1999;33:883-893, incorporated herein by reference in its entirety). 1993;22:854-858).

[0216] Similar studies have demonstrated signs of early arterial disease in both homozygotes and heterozygotes, but individuals with homozygous disease developed symptoms at a much younger age (19 years versus 45 years) and had significantly greater disease severity (see Rubba et al., Metabolism 1990;39:1191-1195, incorporated herein by reference in its entirety).

[0217] Overall, the observations in patients with CBSDH were consistent with previous studies showing that vascular risk increased with Hcy levels (see Boushey et al. JAMA 1995;274:1049-1057; Clarke et al. JAMA 2002;288:2015-2022; Khan et al. Stroke 2008;39:2943-2949, each of which is incorporated herein by reference in its entirety).

[0218] These results suggest that elevated Hcy levels are a risk factor for CV disease. Additionally, lowering Hcy levels significantly reduced the risk of stroke in the general population and in patients with CBSDH.

[0219] Studies have shown that elevated Hcy levels caused by CBSDH can potentially contribute to the development of atherosclerosis and / or thrombosis through various mechanisms. These include molecular events such as the induction of oxidative stress and its downstream effects, such as the activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), a transcription factor that regulates pro-inflammatory and other damage-related genes. Various Hcy-mediated effects that modulate the physiochemical properties of the vascular wall, such as those leading to endothelial dysfunction or arterial stiffness, may contribute to the development of hypertension, thrombosis, or other vascular abnormalities. Finally, there is evidence for direct induction of the coagulation pathway by Hcy, a more direct route leading to thrombosis (Faverzani et al. Cell Mol Neurobiol 2017; Hainsworth et al. Biochim Biophys Acta 2016;1862:1008-1017; Ganguly et al. Nutr J 2015;14:6; Tripathi P. Molecular and biochemical aspects of homocysteine ​​in cardiovascular diseases. International Cardiovascular Forum J 2016;6:13; Fryer et al., each of which is incorporated herein by reference in its entirety). (See, e.g., [Davidson, J., et al.] Arterioscler Thromb 1993;13:1327-1333). After a brief description of the molecular and biochemical mechanisms of atherosclerosis, the following subsections outline potential mechanisms leading to vascular disease in individuals with elevated tHcy levels, including those with CBSDH.

[0220] 2.Atherosclerosis Among the most well-studied conditions leading to thrombosis and resulting vascular occlusion is atherosclerosis, a progressive inflammatory disease affecting the coronary arteries, brain, and peripheral circulation (see Libby et al., Circulation 2005;111:3481-3488, incorporated herein by reference in its entirety). In its early stages, vascular injury leads to endothelial cell (EC) activation, monocyte recruitment to the intima, and macrophage activation. Inflammatory atherosclerotic lesions (fatty streaks) are formed, comprising monocyte-derived, lipid-laden macrophages (foam cells) and T lymphocytes. Progressive lipid accumulation leads to the formation of a lipid core surrounded by a fibrous capsule. During the later stages, activated macrophages secrete enzymes that weaken the fibrous capsule, leading to plaque rupture, hemorrhage, or thrombosis and ischemic stroke / acute coronary syndrome. Plaque rupture exposes tissue factor to the blood in the arterial lumen, allowing it to form a complex with coagulation factors VII / VIIa. This process initiates the coagulation cascade, leading to thrombus formation. Disrupted plaques can lead to mural thrombosis or occlusive thrombosis, which can cause partial or complete occlusion, respectively. Mural thrombosis causes ischemic symptoms such as unstable angina, while occlusive thrombosis leads to acute coronary events such as MI and stroke. Cytokines are involved in all stages of atherosclerosis and have a profound impact on its pathogenesis (see Ramji et al., Cytokine Growth Factor Rev 2015;26:673-685, incorporated herein by reference in its entirety). In addition to being secondary to atherosclerosis, thrombosis can also be activated in the absence of plaque formation, for example, as a result of atrial fibrillation or by direct activation of the coagulation cascade.

[0221] 3. Oxidative stress Studies in patients with elevated Hcy levels have shown that treated and especially untreated patients are susceptible to oxidative stress, as evidenced by altered biomarkers reflecting lipid, protein, and DNA oxidative damage in various tissues (see Vanzin et al. Mol Genet Metab 2011;104:112-117; Vanzin et al. Gene 2014;539:270-274; Vanzin et al. Cell Mol Neurobiol 2015;35:899-911, each of which is incorporated herein by reference in its entirety). Oxidative stress, defined as an imbalance in redox homeostasis, plays a key role in vascular pathologies such as atherosclerosis and its associated thrombosis, which involve oxidative modification of low-density lipoproteins, endothelial activation, and the initiation of vascular inflammatory responses (see Nowak et al., Arterioscler Thromb Vasc Biol 2017;37:e41-e52, incorporated herein by reference in its entirety). Oxidative stress is caused by elevated ROS (e.g., superoxide (O)). 2- ) and hydroxyl (HO - (H2O2)) levels and / or decreased tissue antioxidant (e.g., superoxide dismutase, catalase, and glutathione peroxidase) levels (Faverzani et al., Cell Physiology, Vol. 1, pp. 111-114, 2002, both of which are incorporated herein by reference in their entireties). (See Mol Neurobiol 2017; Nowak et al. Arterioscler Thromb Vasc Biol 2017;37:e41-e52.) In healthy individuals, ROS are generated as a by-product of normal oxidative metabolism. However, in addition, ROS generation is induced by CV risk factors such as cigarette smoke, alcohol consumption, hypercholesterolemia, hypertension, diabetes, and elevated Hcy levels.

[0222] At the molecular level, there are numerous ways in which Hcy can cause increased oxidative stress, some of which have been discussed previously. For example, the accumulation of immunogenic homocysteinylated proteins in the vascular wall can lead to inflammation by activated phagocytes and consequently the production of ROS (O 2- Another potential mechanism is Hcy-induced activation of NMDA receptors, which triggers signaling pathways leading to ROS generation. In cardiac microvascular ECs, Hcy induces the highest levels of O2, especially in activated cells. 2- Hcy induced elevated levels of NADPH oxidase, a cell surface enzyme that produces hydroxybenzoates (NO). A recent study (see Chen et al., Sci Rep. 2017 Jul 31;7(1):6932, incorporated herein by reference in its entirety) suggested that Hcy induces mitochondrial dysfunction in the ischemic rat brain, a likely consequence of increased ROS production. Hcy is also thought to reduce the bioavailability of the beneficial vasodilator NO. 2- Hcy reacts with NO to give the reactive nitrogen species peroxynitrite, and indeed, Hcy-induced increases in tyrosine nitration, an indicator of peroxynitrite-induced protein damage, have also been reported (see Tyagi et al. Vasc Health Risk Manag 2011;7:31-35, incorporated herein by reference in its entirety). More broadly, thiol-thiol interactions involving Hcy are expected to perturb cellular redox status, potentially reducing, for example, the availability of reduced glutathione and impairing protein assembly and folding.

[0223] In a study of CBSDH patients before and after treatment, pyridoxine, folic acid, betaine, and vitamin B 12Treatment with the supplement attenuated lipid oxidative damage in patients but did not alter sulfhydryl content or total antioxidant status, both indicators of tissue antioxidant capacity. Nevertheless, there was a significant negative correlation between sulfhydryl group content and Hcy levels, and a positive correlation between levels of the lipid peroxidation product malondialdehyde and Hcy levels. This suggested a potential mechanistic role for Hcy in the oxidative damage observed in CBSDH (Vanzin et al., Mol. Chem. Soc. 2014, 144:111-112, which is incorporated herein by reference in its entirety). (See Genet Metab 2011;104:112-117). Altered lipid profiles, particularly reduced levels of high-density lipoprotein and enrichment of pro-inflammatory lipid species, were observed in the plasma of untreated and treated CBSDH patients (See Vanzin et al. Cell Mol Neurobiol 2015;35:899-911, incorporated herein by reference in its entirety). Another study reported significantly more DNA damage in CBSDH patients than in healthy individuals (See Vanzin et al. Gene 2014;539:270-274, incorporated herein by reference in its entirety). Collectively, these findings implicate oxidative stress in the pathogenesis of vascular damage associated with elevated Hcy levels. No correlation was found between Met levels and any oxidative stress-related parameters, suggesting that Met and its derivatives contribute little to oxidative damage in CBSDH (Vanzin et al., Mol Genet Metab, 2002, 144:1457-1463, which is incorporated herein by reference in its entirety). 2011;104:112-117).

[0224] Among numerous other molecular effects, oxidative stress has been associated with the activation of NF-κB, a group of transcription factors that regulate the expression of pro-inflammatory genes, such as cytokines, known to be involved in the initiation and progression of atherosclerosis and thrombosis (see Rodriguez-Ayala et al., Atherosclerosis 2005;180:333-340, incorporated herein in its entirety). In vitro studies have shown that treatment of ECs with Hcy activates NF-κB via ROS generation (see van Guldener et al., Curr Hypertens Rep 2003;5:26-31, incorporated herein in its entirety). In addition to modulating gene expression, chemical modifications of cellular macromolecules by oxidative stress may directly affect the structure and function of the vasculature and have other local or systemic effects, as discussed in the remainder of this section.

[0225] 4. Changes in blood vessel walls Endothelial dysfunction is typically defined as an imbalance between endothelium-associated factors that modulate vascular contractility and relaxation. Among these factors, NO or "endothelium-derived relaxing factor" is the best known, and hydrogen sulfide (HS) is another that has been recently described (see Jiang et al., Arterioscler Thromb Vasc Biol 2005;25:2515-2521, incorporated herein in its entirety). Several in vitro studies have investigated the effects of Hcy on endothelial function, albeit using very high levels (Jiang et al., Arterioscler Thromb Vasc Biol 2005;25:2515-2521, incorporated herein in their entirety). al.) Arterioscler Thromb Vasc Biol 2005;25:2515-2521; Hossain et al. (J Biol) Chem 2003;278:30317-30327; Cai et al. Blood 2000;96:2140-2148; Zhang et al. J Biol Chem 2001;276:35867-35874). One such study reported the unfolded protein response and programmed cell death in human umbilical vein endothelial cells (HUVECs) treated with Hcy, although the Hcy concentrations were several-fold higher than those observed in patients with severely elevated Hcy levels (Zhang et al. J Biol Chem 2001;276:35867-35874, incorporated herein by reference in its entirety). Another report found that Cth gamma lyase (CGL), an enzyme involved in Cth metabolism, produced excess H2S in patients with elevated Hcy levels (Papapetropoulos et al., Proc. J. Med. 2012, 144:111-112, each of which is incorporated herein by reference in its entirety). Natl Acad Sci USA 2009;106:21972-21977;Szabo C et al.Br J Pharmacol 2011;164:853-865;Chiku et al.J Biol Chem 2009;284:11601-11612). This observation was significant because elevated H2S levels have been reported to significantly increase collateral vessel growth, capillary density, and local tissue blood flow (Wang et al., Antioxid Redox Signal 2010;12:1065-1077, incorporated herein in its entirety). However, high Hcy levels (0.002-2 mM) did not significantly affect EC proliferation or phospho-eNOS levels in vitro (Saha et al., Cystathionine beta-synthase regulates endothelial function via protein S-sulfhydration. FASEB J 2016;30:441-456, incorporated herein in its entirety). Overall, these results suggest a possible role for Hcy in endothelial dysfunction, although it is still unclear whether Hcy has a direct effect on ECs in vivo due to the high Hcy levels examined in vitro.

[0226] Recent pharmacological and genetic studies have shown that loss of CBS function in ECs is associated with a 50% decrease in cellular HS and a 400% decrease in glutathione, along with a concomitant increase in cellular ROS levels (see Saha et al., FASEB J 2016;30:441-456, incorporated herein by reference in its entirety). Silencing CBS in ECs impaired the phenotypic and signaling responses to vascular endothelial growth factor (VEGF), an effect exacerbated by reduced transcription of vascular endothelial growth factor receptor-2 (VEGFR-2) and neuropilin-1 (NRP-1), key receptors regulating endothelial functions such as angiogenesis. Transcriptional downregulation of VEGFR-2 and NRP-1 was mediated by decreased stability of the transcription factor specific protein 1 (Sp1), a sulfhydrylation target of HS. Supplementation of CBS-silenced ECs with HS, but not glutathione, restored Sp1 levels and Sp1 binding to the VEGFR-2 promoter, while simultaneously increasing VEGFR-2 and NRP-1 expression and VEGF-dependent cell proliferation and migration. This suggests that CBS-mediated protein S-sulfhydrylation is important for maintaining vascular health and function, supporting previous observations that CBSDH patients exhibit endothelial dysfunction (see Celermajer et al. J Am Coll Cardiol 1993;22:854-858; Rubba et al. Metabolism 1990;39:1191-1195, both of which are incorporated herein by reference in their entireties), and raises the possibility that CBSDH patients suffer from additional and distinct types of vascular damage in addition to those observed in individuals with elevated Hcy levels due to other causes.

[0227] Dysregulation of endothelial function or other effects on vasoconstriction can lead to blood pressure (BP) abnormalities. Plasma Hcy levels are directly related to BP, and lowering Hcy with folic acid has been associated with lower BP (see Tripathi P., Molecular and biochemical aspects of homocysteine ​​in cardiovascular diseases. International Cardiovascular Forum J 2016;6:13; Hackam et al., JAMA 2003;290:932-940, both of which are incorporated herein by reference in their entireties). Although the mechanisms leading to these effects remain unclear, Hcy levels are more strongly associated with systolic than diastolic BP, suggesting that elevated Hcy levels increase arterial stiffness. The degree of arterial stiffness is primarily determined by the number and function of smooth muscle cells (SMCs), the collagen:elastin ratio in the ECM, the quality of collagen, and endothelial function (Tripathi P., Molecular and Biochemical Aspects of Homocysteine ​​in Cardiovascular Disease, incorporated herein in its entirety). aspects of homocysteine ​​in cardiovascular diseases).See International Cardiovascular Forum J 2016;6:13).

[0228] Potentially, high levels of Hcy are associated with increased arterial stiffness due to increased SMC proliferation, collagen production, and elastin fiber formation (see van Guldener et al., Curr Hypertens Rep 2003;5:26-31, incorporated herein by reference in its entirety). However, it is also possible that Hcy reduces arterial stiffness by impairing collagen crosslinking. In a study performed in minipigs, diet-induced elevation of Hcy led to a "megaartery syndrome," characterized by hyperpulsatile arteries, systolic (but not diastolic) hypertension, and prolonged reactive hyperemia of conduit arteries due to aortic dilation (see van Guldener et al., Homocysteine ​​and blood pressure. Curr Hypertens Rep 2003;5:26-31, incorporated herein by reference in its entirety). There was also disruption of the elastic lamina of the arterial wall, which correlated with aortic stiffness.

[0229] Consistent with these findings, studies performed in mice with and without CBSDH showed that the cross-sectional area of ​​the blood vessel wall was significantly greater in CBS+ / + mice fed a control diet than in CBS+ / + mice (324 ± 18 μm 2 ) mice fed a control diet compared with CBS+ / - mice (437 ± 22 μM 2 ) and CBS+ / + (442±36μm 2 ) and CBS+ / -(471±46μm 2 ) mice (p<0.05) (see Baumbach et al., Structure of cerebral arterioles in cystathionine beta-synthase-deficient mice. Circ Res 2002;91:931-937, incorporated herein by reference in its entirety).

[0230] During maximal vasodilation, the stress-strain curves of cerebral arterioles from CBS+ / - mice on a control diet and from CBS+ / + and CBS+ / - mice on a high-Met diet were shifted to the right of the curve for CBS+ / + mice on a control diet. This indicated that cerebral arteriolar distensibility was greater in mice with elevated plasma tHcy levels. These results suggest that elevated Hcy levels induce cerebrovascular hypertrophy and alter cerebrovascular mechanics, both effects potentially contributing to an increased incidence of thrombosis, such as stroke, even in the absence of atherosclerosis (see Baumbach et al., Circ Res 2002;91:931-937, incorporated herein by reference in its entirety).

[0231] Further support for the effect of elevated Hcy on vasodilation comes from a study of five Italian patients with CBSDH and tHcy levels ranging from 193.6 to 342 μmol / L (see Evangelisti et al. Int J Cardiol 2009;134:251-254, incorporated herein by reference in its entirety). The patients presented with mild cardiac valve prolapse and / or regurgitation and signs of connective tissue overgrowth.

[0232] 5. Thrombosis Although multiple studies have failed to find an association with the risk of MI, elevated Hcy levels have been associated with a higher risk of deep vein thrombosis, cerebral venous sinus thrombosis, and retinal vein thrombosis (see Spence JD. Lancet Neurol. 2007 Sept;6(9):830-8, incorporated herein by reference in its entirety). Consistent with these results, additional small studies suggest that CBSDH is associated with thrombosis, but not necessarily with atherosclerosis. Vascular imaging of patients with familial hypercholesterolemia (FH) and CBSDH has shown that patients with FH exhibit diffuse and focal thickening of the carotid arteries and endothelial dysfunction, leading to reduced blood flow, whereas patients with CBSDH have plaques in their carotid arteries that are similar to healthy controls in terms of both intima-media thickness (IMT) and blood flow velocity in the middle cerebral artery (see Rubba et al., Stroke 1994;25:943-950, incorporated herein by reference in its entirety). This study suggests that typical atherosclerotic lesions do not require a thrombotic event to precede a thrombotic event in CBSDH, and that medial damage leading to thrombosis can also be caused by arterial dilation.

[0233] Support for this observation comes from a study comparing the prevalence of carotid and femoral atherosclerosis (determined by IMT and ankle-brachial index) in 13 patients with enzymatically proven heterozygous CBSDH compared with 12 healthy subjects with normal Met stress test results (see Valk et al., Stroke 1996;27:1134-1136, incorporated herein by reference in its entirety). No significant differences in mean IMT values, IMT frequency distribution, or IMT in each of the five arterial segments were observed between the groups. These results could be explained by the fact that the heterozygous individuals were too young (all under 50 years of age) to develop structural vascular changes. However, these data also suggest that elevated Hcy levels may primarily affect the coagulation cascade, at least in younger patients. Indeed, a case report of three unrelated CBSDH patients found that one patient experienced a stroke due to intraluminal thrombosis and another experienced cardiac or arterial thromboembolism without evidence of head and neck atherosclerosis (see Kelly et al. Neurology 2003;60:275-279, incorporated herein by reference in its entirety).

[0234] Consistent with these observations, addition of Hcy to HUVECs and CV1 ECs irreversibly inactivated the anticoagulants, protein C and thrombomodulin (see Lentz et al., J Clin Invest 1991;88:1906-1914, incorporated herein in its entirety). Moreover, addition of Hcy to cultured human ECs increased procoagulant tissue factor activity in a time- and concentration-dependent manner (see Fryer et al., Arterioscler Thromb 1993;13:1327-1333, incorporated herein in its entirety).

[0235] In both studies, Hcy enhanced the coagulation pathway through a mechanism involving its free thiol groups. Collectively, these data support the hypothesis that perturbations in vascular coagulant mechanisms contribute to increased vascular risk in patients with CBSDH and that this may play an early role in patients with CBSDH, before the effects of Hcy on atherosclerosis become apparent.

[0236] Reduction of plasma Hcy levels reduces the risk of vascular complications, especially stroke, in patients with CBSDH and the general population Recent analyses have found a strong association in the general population between elevated tHcy and stroke risk (see Saposnik et al. Stroke 2009;40:1365-1372; Spence JD. Homocysteine ​​lowering for stroke prevention: Unravelling the complexity of the evidence. Int J Stroke. 2016 Oct;11(7):744-7; Hankey et al. Lancet Neurol 2012;11:512-520, each of which is incorporated herein by reference in its entirety).

[0237] Previously, some studies have shown a reduction in vascular risk (Yap et al., Arteriosclerosis, 2004; Yap ... Thromb Vasc Biol 2001;21:2080-2085; Wilcken et al. J Inherit Metab Dis 1997;20:295-300; Yap et al. Semin Thromb Hemost 2000;26:335-340; Saposnik et al. Stroke 2009;40:1365-1372; Huo et al. JAMA 2015;313:1325-1335; Hankey et al. Lancet Neurol 2012;11:512-520), others showed no benefit (Marti-Carvajal et al. Cochrane Database Syst Rev, each of which is incorporated herein by reference in its entirety). 2015;1:CD006612; Ebbing et al. JAMA 2008;300:795-804; Bonaa et al. N Engl J Med 2006;354:1578-1588; Liem et al. Heart 2005;91:1213-1214; Toole et al. JAMA 2004;291:565-575; The Vitamins to Prevent Stroke (VITATOPS) trial: B vitamins in patients with recent transient ischemic attack or stroke in a randomized, double-blind, parallel, placebo-controlled trial in the VITAmins TO Prevent Stroke (VITATOPS)trial: a randomized, double-blind, parallel, placebo-controlled trial)(Lancet Neurol 2010;9:855-865; Albert et al. JAMA 2008;299:2027-2036), the benefits of Hcy-lowering interventions in patients with elevated Hcy levels appeared mixed. Many of these trials focused on vitamin interventions for Hcy lowering, primarily B vitamins (B6 and B7). 12 ) and folic acid supplementation. Results of the study included whether subjects lived in areas where folic acid fortification was practiced, whether they had renal dysfunction that made them more vulnerable to cobalamin toxicity, and whether they had vitamin B deficiency, which is associated with malabsorption relatively common in the elderly. 12 These analyses were subject to confounding factors, such as whether or not the patient had a deficiency or was receiving antiplatelet medication. These recent analyses considered these confounding factors and concluded that elevated tHcy increases the risk of stroke in the general population when accounting for such important variables (see Mudd et al., Arterioscler Thromb Vasc Biol 2000;20:1704-1706; Spence JD., Int J Stroke. 2016 Oct;11(7):744-7; Spence JD, Clin Chem Lab Med. 2013 Mar 1;51(3):633-7, each of which is incorporated herein by reference in its entirety).

[0238] Hcy-lowering therapy reduces the risk of stroke, even in individuals without CBSDH. In the HOPE-2 (Cardiac Outcomes Prevention Evaluation 2) trial, 5522 adults aged 55 years or older with a history of vascular disease or diabetes and at least one additional CV risk factor were treated with vitamin supplementation (folic acid, vitamin B6, and vitamin B) for 5 years. 12 ) or placebo (Saposnik et al., incorporated herein by reference in its entirety). (See [link removed]. et al.) Stroke 2009;40:1365-1372). The mean baseline Hcy concentration was 11.5 μmol / L in both groups, and patients taking daily vitamin supplements containing more than 0.2 mg of folic acid at baseline were excluded from the study. Overall, lower Hcy (mean 3.0 μmol / L vs. placebo) was associated with a significant 27% relative risk reduction (1.3% absolute reduction) in stroke (HR, 0.75; 95% CI, 0.59-0.97) and nonsignificant reductions in ischemic stroke (HR, 0.81; 95% CI, 0.60-1.09) and hemorrhagic stroke (HR, 0.80; 95% CI, 0.32-2.02). In subgroup analyses, the greatest relative risk reduction in stroke (4.3% absolute risk reduction) was observed among patients with baseline Hcy levels in the highest quartile. The treatment benefit was greatest in patients younger than 69 years, in areas without dietary folic acid fortification, and in patients not receiving antiplatelet or lipid-lowering medications at enrollment. Thus, the HOPE-2 trial reported a reduction in the incidence of stroke in individuals receiving B vitamins versus placebo (hazard ratio (HR) 0.75; 95% CI, 0.59-0.97), although the risk of MI was similar in both treatment groups (RR 0.98; 95% CI, 0.85-1.14) (Saposnik et al. Stroke 2009;40:1365-1372; Lonn E, Yousuf, both of which are incorporated herein by reference in their entireties). Yusuf S, Arnold MJ et al. Homocysteine ​​lowering with folic acid and B vitamins in vascular disease. N Engl J Med 2006;354:1567-1577).

[0239] Consistent with the results from the HOPE-2 trial, a subanalysis of the VITATOPS trial, which randomized 8,164 patients with a recent stroke or transient ischemic attack to double-blind treatment with B vitamins or placebo over a median period of 3.4 years, found that B vitamins significantly reduced the primary composite outcome (stroke, MI, or death from vascular causes) among patients not receiving antiplatelet therapy at baseline (17% vs. 21% with placebo; HR 0.76, 0.60-0.96). No significant effect of B vitamins was observed in individuals receiving antiplatelet therapy (Hankey et al., "Antiplatelet therapy and the effects of B vitamins in patients with a previous stroke or transient ischemic attack: a post-hoc subanalysis of the VITATOPS randomized, placebo-controlled trial," incorporated herein by reference in its entirety). of B vitamins in patients with previous Stroke or transient ischemic attack: a post-hoc sub-analysis of VITATOPS, a randomized, placebo-controlled trial. Lancet Neurol 2012;11:512-520). In this study, tHcy levels significantly decreased from 12.4-13.7 μmol / L at baseline to 9.9-10.5 μmol / L after vitamin therapy, regardless of whether patients received antiplatelet therapy (p<0.0001 for both treatment groups) (Hankey et al., Lancet Neurol 2012;11:512-520, incorporated herein by reference in its entirety).

[0240] The inflammatory cascade is believed to contribute to the pathogenesis of ischemic stroke. A report from the Framingham Offspring Study in 3224 participants (see Shoamanesh et al., Neurology. 2016 September;87(12):1206-11, which is incorporated herein by reference in its entirety) found that elevated levels of tHcy and three other inflammatory markers were strongly associated with the risk of ischemic stroke and improved the predictive ability of the Framingham Stroke Risk Profile score.

[0241] A meta-analysis of data from the VISP and VITATOPS studies found that patients with normal renal function who had not previously been exposed to high-dose cyanocobalamin significantly benefited from vitamin therapy containing high-dose cyanocobalamin (RR 0.78, 0.67 to 0.90; interaction p=0.03), but that vitamin therapy containing high-dose cyanocobalamin (a form of vitamin B) had no effect on stroke risk in individuals with impaired renal function (RR 1.04, 95% CI 0.84 to 1.27) (see Spence JD. Lancet Neurol. 2007 Sep;6(9):830-8, incorporated herein by reference in its entirety). These results suggested a potentially confounding effect of cyanocobalamin in patients with significant renal impairment, known as nephrotoxicity, associated with cyanide accumulation (see Spence JD, Clin Chem Lab Med. 2013 Mar 1;51(3):633-7, incorporated herein by reference in their entireties). Consistently, in the DIVINe trial (Diabetes Intervention with Vitamins in Nephropathy), high-dose B vitamins, including 1000 μg cyanocobalamin, were harmful and exacerbated eGFR decline (see Spence JD. Int J Stroke. 2016 Oct;11(7):744-7; House et al. JAMA 2010;303:1603-1609, both incorporated herein by reference in their entireties). Taken together, these findings support the use of non-cyanide-containing B vitamins, such as methylcobalamin, instead of cyanocobalamin to reduce Hcy levels in individuals at high risk of stroke, particularly those with renal failure.

[0242] Cyanocobalamin and cyanide toxicity were further implicated in earlier trials as confounding factors in the presence of renal impairment by results from the Chinese Stroke Primary Prevention Trial (CSPPT) (see Huo et al. JAMA. 2015 Apr. 7;313(13):1325-35, incorporated herein by reference in its entirety). The treatment benefit demonstrated in renal impairment in the CSPPT was in direct contrast to the lack of benefit observed in the DIVINe, VISP, and VITOPS trials, likely due to cyanocobalamin treatment in later trials.

[0243] Because folic acid fortification had not yet been implemented in China, we were able to study the effect of folic acid supplementation on reducing tHcy levels in a large population. The CSPPT, a randomized, double-blind trial conducted in 20,702 adults with hypertension but no history of stroke or MI, demonstrated that folic acid significantly reduced the risk of first stroke (2.7% vs. 3.4% without folic acid, HR 0.79; 95% CI 0.68-0.93), first ischemic stroke (2.2% vs. 2.8% without folic acid, HR 0.76; 95% CI 0.64-0.91), and composite CV events (CV death, MI, and stroke; 3.1% vs. 3.9% without folic acid, HR 0.80; 95% CI 0.69-0.92). In contrast, there were no significant differences in the risk of hemorrhagic stroke, all-cause mortality, or frequency of AEs between the two groups. A substudy of the CSPPT (see Xu et al. JAMA Intern Med. 2016 Oct 1;176(10):1443-1450, incorporated herein by reference in its entirety) showed that renal impairment (60 mL / min / 1.73 m 2 Both subjects with and without eGFR < 0.01 benefited from folic acid, and a substudy further confirmed that the folic acid-treated group had a much greater drop in serum Hcy than the group not receiving folic acid (1.9 vs. 0.2 μmol / L, respectively, p<0.001).

[0244] A 2017 Cochrane review (see Marti-Carvajal et al. Cochrane Database Syst Rev. 2017 Aug 17;8:CD006612, incorporated herein by reference in its entirety), analyzing 15 randomized controlled trials involving 74,422 participants, reported small differences in the effect of Hcy lowering with B vitamins on stroke, but no effect on MI, death from any cause, or AEs. Compared with placebo / standard care, Hcy-lowering interventions were associated with a reduced incidence of nonfatal or fatal stroke in the general population (4.33% versus 5.1% for control; RR 0.90, 95% CI 0.82 to 0.99), but had no effect on the incidence of nonfatal or fatal MI (7.1% versus 6.0% for placebo; relative risk (RR) 1.02, 95% CI 0.95 to 1.10) or death from any cause (11.7% versus 12.3% for placebo; RR 1.01, 95% CI 0.96 to 1.06). This review was the latest of three previous versions (2009, 2013, and 2015) that had previously concluded there was no evidence supporting the effect of Hcy-lowering interventions on CV events, although the 2015 review pointed to a non-significant trend in reduced incidence of stroke (see Marti-Carvajal Cochrane Database Syst Rev 2015;1:CD006612, incorporated herein by reference in its entirety). As additional trials have become available, the strength of the evidence for Hcy-lowering interventions for stroke has increased.

[0245] The studies included in this review used various regimens of vitamin supplementation as Hcy-lowering therapy (see Marti-Carvajal et al., Cochrane Database Syst Rev. 2017 Aug. 17;8:CD006612, incorporated herein by reference in its entirety). The 2017 review added three new trials to those in the 2015 review. Of the total, 10 trials included pyridoxine plus vitamin B9 (folic acid) and B 121 used folic acid, 5 used vitamin B9 alone, and 1 of 10 used 5-methyltetrahydrofolate instead of folic acid. Some trials included concomitant medications (in both the control and vitamin-treated groups), 7 included lipid-lowering medications, and 1 included an antihypertensive agent. Overall, Hcy-lowering treatments resulted in relatively small reductions in mean tHcy levels. Furthermore, 3 studies were conducted in folic acid-fortified populations, and 2 were conducted in mixed populations (some subjects received a fortified diet and others did not), which may have masked the Hcy-lowering effect. Considering the potential confounding factors affecting such studies, it is not surprising that previous reviews did not identify significant CV effects of vitamin treatment regimens and that the most recent review reported only modest effects on stroke. It is also noteworthy that many potential causes of elevated Hcy levels were not investigated in the studies included in the Cochrane review (see Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612, incorporated herein by reference in its entirety). There may be additional factors that influence the pathophysiology of vascular risk in patients with CBSDH, who are younger than the general population. It is noteworthy that even modest reductions in Hcy levels are significantly associated with reduced stroke risk.

[0246] An interesting question arising from some of these analyses is why some studies have shown an effect of Hcy levels on stroke but not on MI. In relation to this question, Spence (see Spence JD. Lancet Neurol. 2007 Sept;6(9):830-8, incorporated herein by reference in its entirety) pointed out an important difference between MI and cerebral infarction in that cerebral infarction is closely associated with thrombosis / embolic events, whereas in situ thrombosis is secondary to coronary plaque rupture in nearly all MI events. Thus, Spence concluded that a substantial proportion of strokes are associated with thrombotic processes that may be associated with elevated tHcy.

[0247] Elevated tHcy levels may be important not only in cardiogenic cerebral embolism, but also in atherosclerotic embolism and lacunar infarction. A study in elderly patients with atrial fibrillation treated with anticoagulation therapy found that high levels of tHcy (above the 90th percentile) were associated with a 4.7-fold increase in ischemic complications (see Poli et al. J Am Coll Cardiol 2009; 54:999-1002, incorporated herein by reference in its entirety). Another study in patients with cryptogenic ischemic stroke found that patients with patent foramen ovale (a risk factor for cerebral infarction) had significantly higher plasma tHcy levels than those without (8.9±3 vs. 7.9±2.6 μmol / L, respectively; p=0.021) (see Ozdemir et al. J Neurol Sci 2008;275:121-127, incorporated herein by reference in its entirety). In a review of these findings, Spence (see Spence JD. Homocysteine ​​lowering for stroke prevention: Unravelling the complexity of the evidence. Int J Stroke. 2016 Oct;11(7):744-7, incorporated herein in its entirety) suggested that although Hcy primarily influences the formation of red thrombi (fibrin polymer meshes formed by trapped red blood cells in the setting of stasis), tHcy levels were also significantly higher in patients with microemboli on transcranial Doppler (16.2 vs. 10.1 mmol / L), suggesting that lacunar infarcts and carotid plaques are also significantly associated with tHcy, and that most such microemboli are likely to be platelet aggregates, which are reduced by dual antiplatelet therapy (Spence JD. Homocysteine ​​lowering for stroke prevention: Unravelling the complexity of the evidence. Int J Stroke. 2016 Oct;11(7):744-7, incorporated herein in its entirety). JD.) Homocysteine ​​lowering for stroke prevention: Unravelling the complexity of the evidence. Int J Stroke. 2016 October;11(7):744-7.

[0248] A study of 32 CBSDH patients (aged 9-66 years) treated with pyridoxine, folic acid, and hydrocobalamin for a total of 539 patient-years reported two vascular events (one fatal pulmonary embolism and one MI) during treatment (see Wilcken et al. J Inherit Metab Dis 1997;20:295-300, incorporated herein by reference in its entirety). In an epidemiological study by Mudd et al., 21 events would have been expected over the same period without treatment (RR 0.09 (95% CI 0.02-0.38); p=0.0001). A second study conducted in 84 patients from three countries, between 2.5 and 70 years of age, reported five cases of venous embolism (VE) during 1314 patient-treatment years: one pulmonary embolism, two MI, and two abdominal aortic aneurysms (see Yap et al., Semin Thromb Hemost 2000;26:335-340, incorporated herein by reference in its entirety). Mudd et al. (see Mudd et al., Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety) found that in untreated patients, 53 cases of VE were expected (RR 0.091 (95% CI 0.043-0.190); p<0.001).

[0249] Supporting evidence for these results comes from a large, international, multicenter, observational study of 158 patients with CBSDH, most of whom were in the 10-30 year age range (see Yap et al., Arterioscler Thromb Vasc Biol 2001;21:2080-2085, incorporated herein by reference in its entirety). In this study, 17 vascular events were observed among 12 treated individuals: 3 pulmonary embolisms, 2 MIs, 5 deep vein thrombosis, 3 cerebrovascular accidents, 1 transient ischemic attack, 1 sagittal sinus thrombosis, and 2 abdominal aortic aneurysms. Without treatment, 112 vascular events would have been expected in a similar population (RR 0.09 (95% CI 0.036-0.228); p<0.0001). This study also highlights how even treated and well-complied young adult CBSDH patients suffer from poor clinical outcomes compared with the general population.

[0250] Elevated plasma tHcy levels are a risk factor for vascular disease and a strong predictor of mortality in CAD patients with and without CBSD (Mudd et al. Am J Hum Genet 1985;37:1-31; Karaca et al. Gene 2014;534:197-203; Kelly et al. Neurology 2003;60:275-279; Faeh et al. Swiss Med Wkly 2006;136:745-756; Boushey et al. JAMA 1995;274:1049-1057; Clarke et al. JAMA 2002;288:2015-2022; Hankey et al., each of which is incorporated herein by reference in its entirety). et al.) Lancet 1999;354:407-413; Khan et al. Stroke 2008;39:2943-2949; Graham et al. JAMA 1997;277:1775-1781; Clarke et al. N Engl J Med 1991;324:1149-1155; Clarke et al. Ir J Med Sci 1992;161:61-65; Woodward et al. Blood Coagul Fibrinolysis 2006;17:1-5; Refsum et al. Annu Rev Med 1998;49:31-62; Yoo et al. Stroke 1998;29:2478-2483; Selsun et al. N Engl J Med 1995;332:286-291; Wald et al. BMJ 2002;325:1202; Bautista et al. J Clin Epidemiol 2002;55:882-887; Brattstrom et al. Atherosclerosis 1990;81:51-60; Lussana et al.(See Thromb Res 2013;132:681-684; Casas et al. Lancet 2005;365:224-232; McCully KS. Am J Pathol 1969;56:111-128; Magner et al. J Inherit Metab Dis 2011;34:33-37; Wilcken et al. J Clin Invest 1976;57:1079-1082; Nygard et al. N Engl J Med 1997;337:230-236).

[0251] Of the evidence for a causal relationship between tHcy levels and CV risk (see Boushey et al. JAMA 1995;274:1049-1057, incorporated herein in its entirety), the strongest relationship has been demonstrated between tHcy and stroke or PAD (see Clarke et al. JAMA 2002;288:2015-2022; Khan et al. Stroke 2008;39:2943-2949; Wald et al. BMJ 2009;10:1062-1064, incorporated herein in its entirety), including in patients with both homozygous and heterozygous CBS mutations (see Rubba et al. Metabolism 1990;39:1191-1195, incorporated herein in their entirety). 2002;325:1202; Casas et al. Lancet 2005;365:224-232; Brattstrom et al. Haemostasis 1989;19 Suppl 1:35-44).

[0252] The mechanisms potentially linking elevated Hcy to vascular injury are varied and complex. Several studies have implicated oxidative stress, including those by NF-κB-mediated inflammatory / immune activation (see Rodriguez-Ayala et al. Atherosclerosis 2005;180:333-340; van Guldener et al. Curr Hypertens Rep 2003;5:26-31, both of which are incorporated herein by reference in their entireties) (Faverzani et al. Cell Mol Neurobiol 2017; Nowak et al. Arterioscler Thromb Vasc Biol 2017;37:e41-e52; Vanzin et al. Mol Genet Metab 2011;104:112-117; Vanzin et al. Gene 2014;539:270-274; Vanzin et al. Lipid, Cell Mol Neurobiol 2015;35:899-911. Although such inflammatory processes are characteristic of atherosclerosis, typical atherosclerotic lesions need not precede thrombotic events in patients with CBSDH (see Rubba et al. Stroke 1994;25:943-950; de Valk et al. Stroke 1996;27:1134-1136, both of which are incorporated herein by reference in their entireties). Indeed, central injury leading to thrombosis is associated with Hcy-mediated endothelial dysfunction (Marti-Carvajal et al. Cochrane Database Syst Rev 2015;1:CD006612; Celermajer et al. J Am Coll Cardiol 1993;22:854-858; Rubba et al. Metabolism 1990;39:1191-1195; Jiang et al. Arterioscler Thromb Vasc Biol 2005;25:2515-2521; Hossain et al. J Biol Chem 2003;278:30317-30327; Cai et al. Blood 2000;96:2140-2148; Zhang et al. J Biol Chem 2001;276:35867-35874; Papapetropoulos et al. Proc Natl Acad Sci USA 2009;106:21972-21977; Szabo et al. Br J Pharmacol 2011;164:853-865; Chiku et al. J Biol Chem 2009;284:11601-11612; Wang et al. Antioxid Redox Signal 2010;12:1065-1077; Saha S, et al. FASEB J 2016;30:441-456; Ebbing et al. JAMA 2008;300:795-804; Bonaa et al. N Engl J Med 2006;354:1578-1588), enhanced coagulation pathways (see Spence JD. Int J Immunol 2011;164:853-865; Chiku et al. J Biol Chem 2009;284:11601-11612; Wang et al. Antioxid Redox Signal 2010;12:1065-1077; Saha S, et al. FASEB J 2016;30:441-456; Ebbing et al. JAMA 2008;300:795-804; Bonaa et al. N Engl J Med 2006;354:1578-1588, each of which is incorporated herein by reference in its entirety), and enhanced coagulation pathways (see Spence JD. Int J Immunol 2011;164:853-865; Chiku et al. J Biol Chem 2009;284:11601-11612; Wang et al. Antioxid Redox Signal 2010;12:1065-1077; Saha S, et al. FASEB J 2016;30:441-456; Ebbing et al. JAMA 2008;300:79 Stroke. 2016 Oct;11(7):744-7; Fryer et al. Arterioscler Thromb 1993;13:1327-1333; Lentz et al. J Clin Invest 1991;88:1906-1914), and increased vasodilation similar to the thrombotic process associated with Marfan patients (Kelly et al. Neurology 2003;60:275-279; Tripathi P., each entirely incorporated herein by reference).) Molecular and biochemical aspects of homocysteine ​​in cardiovascular diseases. International Cardiovascular Forum J 2016;6:13; van Guldener et al. Curr Hypertens Rep 2003;5:26-31; Hackam et al. JAMA 2003;290:932-940; Baumbach et al. CBSDH is thought to be caused by endothelial dysfunction (see Evangelisti et al., Circ Res 2002;91:931-937; Evangelisti et al., Int J Cardiol 2009;134:251-254; de Valk et al., Stroke 1996;27:1134-1136). Consistent with the observation of endothelial dysfunction in patients with CBSDH, pharmacological and genetic research studies have implicated CBS-mediated protein S-sulfhydrylation in the maintenance of vascular health and function (see Celermajer et al., J Am Coll Cardiol 1993;22:854-858; Rubba et al., Metabolism 1990;39:1191-1195, both of which are incorporated herein by reference in their entireties). Thus, the mechanisms of vascular injury in patients with CBSDH may be more variable than in the general population with CV disease.

[0253] There is considerable evidence that lowering Hcy is beneficial for stroke risk in individuals with and without CBSDH (see Saposnik et al. Stroke 2009;40:1365-1372; Huo et al. JAMA 2015;313:1325-1335; Lonn et al. N Engl J Med 2006;354:1567-1577; Hankey et al. Lancet Neurol 2012;11:512-520; Spence JD. Lancet Neurol. 2007 Sep;6(9):830-8, each of which is incorporated herein by reference in its entirety). In studies performed in the general population, where mean baseline Hcy levels were relatively low, reductions in stroke incidence were significantly correlated with Hcy-lowering interventions, pointing to benefits of even small reductions in tHcy levels.

[0254] A recent review of relevant trials supports the beneficial effects of Hcy-lowering treatment in stroke (see Marti-Carvajal et al. Cochrane Database Syst Rev. 2017 Aug. 17;8:CD006612, incorporated herein by reference in its entirety). Folic acid fortification, vitamin B, which may leave patients vulnerable to high-dose cyanocobalamin, 12 Confounding factors, such as whether there was a deficiency or renal insufficiency or whether subjects were receiving antiplatelet medication, obscured trial outcomes. Recent analyses have demonstrated that when such factors are accounted for, the association between tHcy and stroke risk in the general population is robust (Spence, 2004, incorporated herein by reference in its entirety). See Spence JD, Clin Chem Lab Med. 2013 Mar 1;51(3):633-7. Importantly, however, the vascular benefits of Hcy reduction have been consistently demonstrated in patients with CBSDH, who have much higher tHcy levels than the general population (see Non-Patent Document 6; Eslamiyeh et al. Iran J Child Neurol 2015;9:53-57; Saboul et al. J Child Neurol 2015;30:107-112; Yap et al. Arterioscler Thromb Vasc Biol 2001;21:2080-2085; Woods et al. BMJ Case Rep 2017;2017; Wilcken et al. J Inherit Metab Dis 1997;20:295-300; Yap et al. Semin Thromb Hemost 2000;26:335-340; see Ruhoy et al. Pediatr Neurol 2014;50:108-111).

[0255] These findings suggest that elevated Hcy levels are a risk factor for CV disease, especially stroke, in patients with and without CBSDH, and that CV or cerebrovascular risk can be reduced through long-term Hcy-lowering therapy.

[0256] Thromboembolism is a leading cause of morbidity and premature mortality in patients with CBSDH (see Mudd et al. Am J Hum Genet 1985;37:1-31; Karaca et al. Gene 2014;534:197-203; Yap S. J Inherit Metab Dis 2003;26:259-265, each of which is incorporated herein by reference in its entirety). The overall rate of thromboembolic events in patients with untreated CBSDH is approximately 10% per year (see Cattaneo M. Semin Thromb Hemost 2006;32:716-723, entirely incorporated herein by reference), with the risk increasing after surgery and during or immediately after pregnancy (see Mudd et al. Am J Hum Genet 1985;37:1-31; Novy et al. Thromb Haemost 2010;103:871-873, both entirely incorporated herein by reference). Although thromboembolism can affect any blood vessel, venous thrombosis, particularly cerebral venous sinus thrombosis (CSVT), is more common than arterial thrombosis in patients with CBSDH (Mudd et al. Am J Hum Genet 1985;37:1-31; Karaca et al. Gene 2014;534:197-203; Eslamiyeh et al. Iran J Child Neurol 2015;9:53-57; Saboul et al. J Child Neurol 2015;10:10-11; each of which is incorporated herein by reference in its entirety). (See Neurol 2015;30:107-112).

[0257] A study in 629 untreated CBSDH patients found that of 253 observed vascular events (occurring in 158 patients), 81 (32%) were cerebrovascular accidents, 130 (51%) involved peripheral veins (32 of which resulted in pulmonary embolism), 10 (4%) led to myocardial infarction (MI), 28 (11%) involved peripheral arteries, and 4 (2%) did not fall into any of these categories (see Mudd et al. Am J Hum Genet 1985;37:1-31, incorporated herein by reference in its entirety). Cerebrovascular accidents, particularly CSVT, have been described in infants (see Mahale et al., J Pediatr Neurosci. 2017 Apr-June;12(2):206-207, incorporated herein in its entirety), but more typically present in young adults (see Yap et al., Arterioscler Thromb Vasc Biol 2001;21:2080-2085, incorporated herein in its entirety). Cerebrovascular events have been reported to be minimally related to a patient's pyridoxine response category.

[0258] The risk of vascular events is approximately 30% in patients under 20 years of age, rising to 50% by age 30. However, symptoms can occur at any age, and fatal thrombosis has been described in infants as young as 6 months of age (see Cardo et al., Dev Med Child Neurol 1999;41:132-135, incorporated herein by reference in its entirety). After age 10, one vascular event can be expected every 25 years. Generally, the first signs of CBSDH in children are mental retardation, which manifests as developmental delay during the first or second year of life, and / or lens dislocation / high myopia. In contrast, adults are more likely to present with vascular events.

[0259] D. Effect of diet on phenotypic outcomes In some embodiments, I278T mice, a mouse model of HCU, were used to assess the long-term effects of enzyme therapy for HCU using 20NHS PEG-CBS on clinical endpoints relevant to human patients. The efficacy of 20NHS PEG-CBS and MRD alone was assessed in a background of normal methionine intake (REG) and a methionine-restricted diet (MRD). Treatment with 20NHS PEG-CBS can result in a 90% reduction in plasma homocysteine ​​concentrations and correction of learning / cognition, endothelial dysfunction, hemostasis, bone mineralization, and body composition phenotypes associated with HCU. In certain embodiments, treatment with 20NHS PEG-CBS in a background of MRD normalized plasma Hcy. MRD alone was observed to reduce plasma Hcy by 67% and correct the HCU phenotype in I278T mice. However, MRD increased anxiety and reduced bone mineral density in both I278T mice and wild-type controls. Therefore, 20NHS PEG-CBS is highly effective in treating HCU in subjects on a REG or Met-restricted diet. Indeed, ET with 20NHS PEG-CBS on a background of normal Met intake provides comparable or superior results compared to a Met-restricted diet.

[0260] E. Neurological complications Studies have shown that early reduction of Hcy levels induced by a low-Met diet, folic acid / B vitamin supplementation, and / or pyridoxine and betaine therapy can prevent and sometimes reverse the progression of various neurological disorders and allow normal IQ development in CBSDH patients (El Bashir et al. JIMD Rep 2015;21:89-95; Yap et al. J Inherit Metab Dis 2001;24:437-447; Mech AW, Farah A. Correlation of clinical response with homocysteine ​​reduction during therapy with reduced B vitamins in patients with MDD positive for MTHFR C677T or A1298C polymorphisms: a randomized, double-blind, placebo-controlled trial, each of which is incorporated herein by reference in its entirety). MDD who are positive for MTHFR C677T or A1298C polymorphism: a randomized, double-blind, placebo-controlled study). J Clin Psychiatry 2016;77:668-671; Grobe H. Eur J Pediatr 1980;135:199-203.Further evidence that significant reduction, or even normalization, of Hcy levels resulted in complete or partial correction of CNS outcomes is provided by case studies in patients with CBSDH (Yap et al. J Inherit Metab Dis 2001;24:437-447; Brenton et al. J Child Neurol 2014;29:88-92; Rezazadeh et al. Child Neurol Open 2014;1:2329048X14545870; Kaeser et al. J Neurol Neurosurg Psychiatry 1969;32:88-93; Colafrancesco et al., each of which is incorporated herein by reference in its entirety). et al.) Eur J Pediatr 2015;174:1263-1266; Yokoi et al. Pediatr Int 2008;50:694-695; Li et al. Pathology 1999;31:221-224).

[0261] The association between elevated Hcy levels and CNS symptoms, including mental retardation, neurodegenerative disorders, seizures, dystonia, psychosis, cognitive impairment, dementia, and depression, has been well documented in CBSDH patients and the general population (12; Abbott et al. Am J Med Genet 1987;26:959-969; Mudd et al. Disorders of transsulfuration. Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw Hill; 2001;1279-1327; Hidalgo et al. al.) Eur Child Adolesc Psychiatry 2014;23:235-238; Smith et al. PLoS One 2010;5:e12244; Seshadri et al. N Engl J Med 2002;346:476-483; Bottiglieri et al. J Neurol Neurosurg Psychiatry 2000;69:228-232; Bjelland et al. Arch Gen Psychiatry 2003;60:618-626; Tolmunen et al. Am J Clin Nutr 2004;80:1574-1578; Kaeser et al. J Neurol Neurosurg Psychiatry 1969;32:88-93; Golimbet et al. Psychiatry Res 2009;170:168-171; Clarke et al. Arch Neurol 1998;55:1449-1455; Permoda-Osip et al. Neuropsychobiology 2014;69:107-111; Oliveira et al. BMJ Case Rep 2016;2016; Troen et al. Proc Natl Acad Sci USA 2008;105:12474-12479; Sudduth et al. J Cereb Blood Flow Metab 2013;33:708-715; Hainsworth et al. Biochim Biophys Acta 2016;1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011;49:435-441; Kim et al. al. J Nutr 2007;137:2093-2097; Selhub et al. Am J Clin Nutr 2000;71:614S-620S; McCaddon et al. Dement Geriatr Cogn Disord 2001;12:309-313; Smallwood et al. Neuropathol Appl Neurobiol 2012;38:337-343; Beydoun et al. BMC Public Health 2014;14:643; Gortz et al. J Neurol Sci 2004;218:109-114; Health Quality O. Vitamin B12 and cognitive function: an evidence-based analysis analysis). Ont. Health Technol. Assess. Ser. 13(23), 1e45. 2013. Ref Type: Online Source; see Salagre et al. Eur Psychiatry 2017;43:81-91. The mechanism leading to CNS tHcy-mediated neuronal damage in individuals with elevated Hcy levels is Mudd et al. Disorders of transsulfuration. Scriver CL, Beaudet AL, Sly WS, each of which is incorporated herein by reference in its entirety. WS), Valle D (eds.). Metabolic and molecular basis of genetic diseases. Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw Hill; 2001; 1279-1327; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008-1017; Stefanello et al. Metab Brain Dis 2007; 22:172-182; Toborek et al. Atherosclerosis 1995; 115:217-224), vascular endothelial damage caused by Hcy-mediated oxidative stress (see Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004;287:R39-R46), neuronal loss (see Yeganeh et al. J Mol Neurosci 2013;50:551-557; Heider et al. J Neural Transm Suppl 2004;1-13, both of which are incorporated herein by reference in their entireties), and diminished neural network activity (see Gortz et al. J Neurol Sci 2004;218:109-114, both of which are incorporated herein by reference in their entireties). Depression and seizures are thought to be caused, at least in part, by Hcy-mediated decreases in brain adenosine levels and subsequent decreases in norepinephrine and dopamine levels (see Mech et al. J Clin Psychiatry 2016;77:668-671; Domagala et al. Thromb Res 1997;87:411-416; Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004;287:R39-R46; Folstein et al. Am J Psychiatry 2007;164:861-867, each of which is incorporated herein by reference in its entirety).

[0262] These findings demonstrate a strong correlation between Hcy levels and increased risk of CNS damage in patients with CBSDH. Early Hcy-lowering therapy is essential for normal development in children with early-onset CBSDH and for correction or amelioration of CNS damage in patients diagnosed with CBSDH later in life.

[0263] A study of 63 CBSDH patients found that 51% had psychiatric disorders, including anxiety and episodic depression (10%), chronic behavioral disorders (e.g., aggression and drug or alcohol abuse) (17%), chronic obsessive-compulsive disorder (5%), and personality disorders (19%) (see Abbott et al. Am J Med Genet 1987;26:959-969, incorporated herein in its entirety). Psychosis may be a presenting symptom in adolescence (Hidalgo et al. Eur Child Adolescent Psychiatry 1987;26:959-969, incorporated herein in its entirety). 2014;23:235-238).

[0264] When untreated, approximately 90% of pyridoxine non-responders have learning difficulties (see Mudd et al. Am J Hum Genet 1985;37:1-31, incorporated herein in its entirety), and IQs typically range from 10 to 138 in pyridoxine non-responders, averaging 57, compared with 79 in untreated pyridoxine-responders and 105 in well-compliant treated pyridoxine-responders (see Yap et al. J Inherit Metab Dis 2001;24:437-447, incorporated herein in its entirety). Seizures affect 20% of non-responders by age 12 years, and several cases of movement disorders not related to basal ganglia infarction have been reported, including polymyoclonus, dystonia, and Parkinson's disease (see Non-Patent Document 12; Rezazadeh et al., Child Neurol Open 2014;1:2329048X14545870, both of which are incorporated herein by reference in their entireties).

[0265] The association between CBSDH and neuropsychiatric symptoms was first described by Schimke et al. in 1965 (see Schimke et al. JAMA 1965;193:711-719, incorporated herein in its entirety). The association was later supported by studies reporting psychopathology in over 50% of CBS-deficient patients (see Abbott et al. Am J Med Genet 1987;26:959-969, incorporated herein in its entirety). Since then, numerous epidemiological studies have demonstrated a positive dose-dependent relationship between mild elevations in homogeneous plasma tHcy and the risk of CNS disorders, including mental retardation and neurodegenerative diseases (see Non-Patent Document 12; Seshadri et al. N Engl J Med 2002;346:476-483; Clarke et al. Arch Neurol 1998;55:1449-1455; Hainsworth et al. Biochim Biophys Acta 2016;1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011;49:435-441; Kim J et al. J Neurosci 2011;1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011;49:435-441; Kim J et al. J Neurosci 2011;1862:1008-1017; Herrmann et al. J Neurosci 2011;1862:1008-1017 ... Herrmann et al. J Neurosci 2011;1862:1008-1017; Kim J et al. J Nutr 2007;137:2093-2097; Selhub et al. Am J Clin Nutr 2000;71:614S-620S; McCaddon et al. Dement Geriatr Cogn Disord 2001;12:309-313; Smallwood et al. Neuropathol Appl Neurobiol 2012;38:337-343; Beydoun et al. (See Mudd et al., Disorders of Transsulfuration, 2014;14:643.) In general, patients with severely elevated Hcy levels (50-200 μM / L) tend to present with acute neuronal dysfunction, including seizures and psychosis, whereas more moderate Hcy levels (15-50 μM / L) are associated with cognitive impairment and dementia (Mudd et al., Disorders of Transsulfuration, 2014;14:643, both of which are incorporated herein by reference in their entireties). of transsulfuration. Scriver CL, Beaudet AL, Sly WS, Valle D (eds.). The Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw-Hill. Hill); 2001; 1279-1327; see Gortz et al. J Neurol Sci 2004; 218:109-114).

[0266] Elevated Hcy levels are associated with cognitive impairment (see Smith et al., PLoS One 2010;5:e12244; Seshadri et al., N Engl J Med 2002;346:476-483, both of which are incorporated herein by reference in their entireties), the development of dementia (see Health 2010;5:e12244; Seshadri et al., N Engl J Med 2002;346:476-483, both of which are incorporated herein by reference in their entireties), and the development of dementia (see Health 2010;5:e12244; Seshadri et al., N Engl J Med 2002;346:476-483, both of which are incorporated herein by reference in their entireties). Quality O. Vitamin B12 and cognitive function: an evidence-based analysis. Ont. Health Technol. Assess. Ser. 13(23), 1e45. 2013. Ref Type: see online source) and Alzheimer's disease (see Seshadri et al. N Engl J Med 2002;346:476-483, incorporated herein by reference in its entirety). Additionally, elevated Hcy levels (greater than 15 μmol / L) have been shown to be present in up to 90% of patients with depression (see Bottiglieri et al. J Neurol Neurosurg Psychiatry 2000;69:228-232; Bjelland et al. Arch Gen Psychiatry 2003;60:618-626, both of which are incorporated herein by reference in their entireties), and men in the top tertile of tHcy levels are more than twice as likely to suffer from depression than men in the bottom tertile (see Tolmunen et al. Am J Clin Nutr 2004;80:1574-1578, which are incorporated herein by reference in their entireties). Elevated Hcy levels have been implicated in schizophrenia, multiple sclerosis, Parkinson's disease, and fibromyalgia / chronic fatigue syndrome (Kaeser et al., 2002; Ka ... al. J Neurol Neurosurg Psychiatry 1969;32:88-93; Golimbet et al. Psychiatry Res 2009;170:168-171; Clarke et al. It has been commonly reported in cases of episodic dystonia without cerebrovascular disease (see Sinclair et al., Arch Neurol 1998;55:1449-1455) and non-cerebrovascular disease (see Sinclair et al., Mov Disord 2006;21:1780-1782, incorporated herein in its entirety). A possible association between the T833C polymorphism in the CBS gene and bipolar disorder has been described (see Permoda-Osip et al., Neuropsychobiology 2014;69:107-111, incorporated herein in its entirety), and a recent meta-analysis indicated a relationship between elevated Hcy levels and mania / euthymia in individuals with bipolar disorder (see Salagre et al., Eur Psychiatry 2017;43:81-91, incorporated herein in its entirety). The first known case of peripheral neuropathy associated with CBSDH was recently described in an 18-year-old male with CBSDH (Oliveira et al., 2004, incorporated herein by reference in its entirety). al.) BMJ Case Rep 2016;2016), a fatal case of psychosis was previously described in a 17-year-old with undiagnosed CBSDH (see Hidalgo et al. Eur Child Adolesc Psychiatry 2014;23:235-238, incorporated herein by reference in its entirety).

[0267] Direct evidence of the relationship between Hcy levels and dementia comes from animal studies in which Hcy administration was associated with the development of brain lesions (see Troen et al., Proc Natl Acad Sci USA 2008;105:12474-12479; Sudduth et al., J Cereb Blood Flow Metab 2013;33:708-715, each of which is incorporated herein by reference in its entirety). In the first such study, male C57BL6 / J mice with elevated Hcy levels (induced by a vitamin B-deficient diet) had significantly impaired spatial learning and memory, along with significant rarefaction of the hippocampal microvasculature without concomitant gliomas or neurodegeneration (see Troen et al., Proc Natl Acad Sci USA 2008;105:12474-12479, each of which is incorporated herein by reference in its entirety). Total hippocampal capillary length was inversely correlated with Morris water maze escape latency (r = -0.757, p < 0.001) and plasma tHcy (r = -0.631, p < 0.007). Mice fed a Met-enriched diet showed similar, but less pronounced, effects. These findings suggested that elevated Hcy levels are associated with cerebral microvascular rarefaction, leading to cognitive impairment in the absence of or preceding neurodegeneration. This may explain the association between elevated Hcy levels and cognitive decline in humans.

[0268] In the second study, healthy mice were given folic acid, vitamin B6, and B 12These mice were deficient in thyroid hormone (HT) and fed a Met-supplemented diet, which induced moderately elevated Hcy levels (plasma tHcy 82.93 ± 3.56 μmol / L). These mice had spatial memory deficits assessed by a 2-day radial-arm water maze (see Sudduth et al., J Cereb Blood Flow Metab 2013;33:708-715, incorporated herein by reference in its entirety). MRI and histology revealed significant microhemorrhage rates. Neuroinflammation and increased expression and activity of MMP2 and MMP9 (both enzymes implicated in the pathogenesis of cerebral hemorrhage) were also observed. This suggested a link between elevated Hcy levels and vascular dementia, such as Alzheimer's disease.

[0269] In humans, individuals with CBSDH (see El Bashir et al. JIMD Rep 2015;21:89-95; Vatanavicharn et al. J Inherit Metab Dis 2008;31 Suppl 3:477-481; Brenton et al. J Child Neurol 2014;29:88-92; Ruhoy et al. Pediatr Neurol 2014;50:108-111, each of which is incorporated herein by reference in its entirety) and those without CBSDH (see Hogervorst et al. Arch Neurol 2002;59:787-793; Kloppenborg et al. Neurology In both CBSDH and CBSDH (see Vatanavicharn et al. J Inherit Metab Dis 2008;31 Suppl 3:477-481, incorporated herein by reference in its entirety), white matter changes (a sign of vascular damage) are frequently associated with elevated Hcy levels. However, these changes are not necessarily associated with evidence of stroke. In addition, brain imaging studies in patients with CBSDH often show signs of atrophy or venous occlusion (see Vatanavicharn et al. J Inherit Metab Dis 2008;31 Suppl 3:477-481, incorporated herein by reference in its entirety).

[0270] Scans reveal diffuse symmetric abnormal increased signal in the subcortical white matter of the cerebral hemispheres, primarily in the parieto-occipital region, and to a lesser extent, in the deep white matter. 1. Mechanism The exact mechanism by which elevated Hcy levels affect neurological health is unknown. Several animal studies have demonstrated an association between elevated tHcy levels and neurotoxicity and associated neurological and psychiatric disorders.

[0271] Early studies showed that very high intraperitoneal doses of Hcy induced generalized convulsive status epilepticus in rats with cobalt-induced lesions of the motor cortex (see Mudd et al., Disorders of transsulfuration. Scriver CL, Beaudet AL, Sly WS, Valle D, eds., The Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw Hill; 2001; pp. 1279-1327, incorporated herein by reference in its entirety). Seizures were enhanced by the addition of Met and vitamin B, and there was some evidence of synergistic effects of pyridoxal 5'-phosphate and Hcy in blocking postsynaptic γ-aminobutyric acid receptors. Furthermore, treatment of rodent neocortical tissue with Hcy led to adenosine sequestration in the form of AdoHcy (see Heinecke et al. J Biol Chem 1987;262:10098-10103, incorporated herein by reference in its entirety). The authors proposed that adenosine is a predominant inhibitor of brain activity and that the seizure state and psychiatric changes associated with high levels of Hcy may be mediated by a decrease in brain adenosine levels.

[0272] Hcy has been linked to both neurotoxicity and brain morphological changes (see Hainsworth et al., Biochim Biophys Acta 2016;1862:1008-1017, which is incorporated herein by reference in its entirety). For example, studies in rats and rabbits have shown that neuronal damage is caused by Hcy-mediated increases in thiobutyric acid reactive substances (TBARS), an indicator of oxidative stress (see Stefanello et al., Metab Brain Dis 2007;22:172-182; Toborek et al., Atherosclerosis 1995;115:217-224, both of which are incorporated herein by reference in their entirety). Similar increases in plasma TBARS were observed in humans after oral Met loading (see Domagala et al., Thromb Res 1997;87:411-416, incorporated herein in its entirety). Moreover, studies in mouse models of elevated Hcy levels suggested that cellular damage caused by oxidative stress may be enhanced in CBSDH patients because decreased Cys levels result in low levels of neuronal glutathione, an important antioxidant synthesized from glutamate, Cys, and glycine (see Vivitsky et al., Am J Physiol Regul See IntegrComp Physiol 2004;287:R39-R46).

[0273] In vivo Hcy injection into the left ventricle of rat brain produced dose-dependent neuronal loss (see Yeganeh et al., J Mol Neurosci 2013;50:551-557, incorporated herein in its entirety), and incubation of rat midbrain tegmental neurons with Hcy led to fewer, shorter dopaminergic neurites (see Heider et al., J Neural Transm Suppl 2004;1-13, incorporated herein in its entirety). In both studies, the effects of Hcy were blunted by coadministration of Hcy with antagonists of NMDA and metabotropic glutamate receptors, suggesting a glutamate receptor-mediated pathway for Hcy-induced neuronal damage. Further evidence for this pathway comes from a study in which Hcy administration led to dose-dependent lipid peroxidation in rat brain synaptosomes (see Jara-Prado et al. Neurotox Res 2003;5:237-243, incorporated herein by reference in its entirety). Once again, the effect was blocked by administration of an NMDA receptor antagonist.

[0274] Studies in spontaneously active embryonic rat cortical neurons have shown that Hcy levels above the severely elevated Hcy range cause a dose-dependent suppression of neuronal network activity (see Gortz et al., J Neurol Sci 2004;218:109-114, incorporated herein by reference in its entirety). Because such excessive Hcy levels are never reached in patients with CBSDH, the effects observed in this study were not clinically relevant. However, modest increases in homocysteine ​​sulfinic acid and homocysteic acid (oxidized forms of Hcy often seen in patients with elevated Hcy levels) had similar effects. In each case, damage to neural networks was inhibited by 2-amino-5-phosphonovaleric acid, similarly implicating NMDA receptors as a mediator of this Hcy-induced neuronal dysfunction. These results suggest that neuronal dysfunction associated with elevated Hcy levels may be caused by the oxidized forms of Hcy rather than Hcy itself.

[0275] The absence of mental retardation, seizures, and other CNS disorders in patients with Marfan syndrome and other connective tissue disorders suggests that the neurological disorder in CBSDH patients is not caused by a fibrillin or collagen defect (see Mudd et al., Disorders of transsulfuration. Scriver CL, Beaudet AL, Sly WS, Valle D, eds., The Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw Hill; 2001; pp. 1279-1327, incorporated herein by reference in its entirety). In patients with untreated CBSDH, elevated SAM levels and decreased SAH levels inhibit the transmethylation reactions required for myelin synthesis, leading to further neuronal damage (Mudd et al., Disorders of Transsulfuration, vol. 1, pp. 111-114, which are incorporated herein by reference in their entirety). of transsulfuration. Scriver CL, Beaudet AL, Sly WS, Valle D (eds.). The Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw-Hill. Hill; 2001; 1279-1327). Decreased myelin synthesis may also be caused by low serine levels in CBSDH patients due to increased remethylation rates (see Orendac et al. J Inherit Metab Dis 2003; 26:761-773, incorporated herein by reference in their entireties). Finally, Hcy metabolism plays an important role in monoamine synthesis by providing methyl groups for the production of norepinephrine and dopamine (see Mech et al. J Clin Psychiatry 2016; 77:668-671; Folstein et al. Am J Psychiatry 2007; 164:861-867, both incorporated herein by reference in their entireties). Indeed, the "homocysteine ​​hypothesis of depression" (see Folstein et al. Am J Psychiatry 2007;164:861-867, incorporated herein by reference in its entirety) states that elevated Hcy levels, resulting in low levels of norepinephrine and dopamine, are a primary cause of depression.

[0276] Electron microscopy of rat brain biopsies demonstrated structural changes in the cerebral vasculature in animals fed a high-Hcy diet for 8 weeks. These changes were associated with elevated plasma tHcy levels (see Lee et al., J Nutr 2005;135:544-548, incorporated herein by reference in its entirety). An additional 8 weeks of dietary folic acid consumption reduced plasma tHcy levels to normal and significantly reduced the incidence of damaged blood vessels. This suggests that Hcy reduction using folic acid supplementation may reduce the deleterious effects of experimentally induced elevated Hcy levels on the vascular endothelium.

[0277] A study designed to assess the efficacy and safety of reduced B vitamins as monotherapy in adults (N=330) with major depressive disorder (MDD) and CBSDH due to at least one MTHFR polymorphism found that treatment with a combination of reduced B vitamins significantly reduced tHcy levels in 131 treated patients (82.4%) (the mean reduction in this subgroup was 25%; p<0.001), whereas placebo-treated patients demonstrated a small increase in tHcy levels (see Mech et al. J Clin Psychiatry 2016;77:668-671, incorporated herein by reference in its entirety). Treated patients had, on average, a 12-point reduction in the Montgomery-Asberg Depression Rating Scale (MADRS) by week 8, with 42% achieving complete remission (p<0.001). Further clinical improvement correlated with a significant decrease in tHcy levels in the majority of responders. Although this study was not performed in patients with CBSDH, it demonstrated a clear benefit of Hcy lowering in individuals with depression, supporting the "Hcy hypothesis of depression" (see Folstein et al. Am J Psychiatry 2007;164:861-867, incorporated herein by reference in its entirety).

[0278] The benefit of Hcy-lowering therapy in individuals with psychiatric symptoms associated with CBSDH was first demonstrated in a study of 12 late-diagnosed patients (see Grobe H. Eur J Pediatr 1980;135:199-203, incorporated herein by reference in its entirety). Three of these patients were never effectively treated, suffered severe psychological disorders, and died prematurely. The remaining eight patients ranged in age from 1 to 26 years (mean, 13 years), and all had psychiatric symptoms including irritability, ADHD, apathy, and psychosis. Treatment with a low-Met diet using pyridoxine or supplemental L-cysteine ​​for 2 to 9 years was associated with impressive improvements in behavior and intellectual development, correlated with biochemical normalization. The authors emphasized the need to treat all patients, regardless of age at diagnosis and before treatment, due to the reversibility and improvement of CBSDH-associated sequelae observed in the study.

[0279] Using more recent data from an Irish screening program, the mental performance of 23 pyridoxine-nonresponsive individuals with CBSDH (339 patient-treatment years) was compared with that of 10 unaffected sibling controls (see Yap et al. J Inherit Metab Dis 2001;24:437-447, incorporated herein by reference in its entirety). Of the 23 patients identified, 19 were diagnosed with CBSDH through NBS and treated early in life (within 6 weeks of age), 2 were detected later (at ages 2.2 and 2.9 years), and 2 were untreated at the time of evaluation. All patients were treated with a Met-free, cysteine-supplemented synthetic amino acid mixture, vitamin B12, as needed. 12 and folic acid supplements. Early treated patients with poor dietary compliance and all patients with later detected CBSDH used betaine in the last 5 years as an adjunct to treatment.

[0280] Overall, 13 of 19 patients in the early treatment group (mean age 14.4 years; range 4.4-24.9 years) adhered to treatment (defined by a lifetime plasma fHcy median of less than 11 μmol / L) and had no complications, whereas the remaining 6 patients who were non-adherent (mean age 19.9 years; range 13.8-25.5 years) developed complications. The mean full-scale IQ (FIQ) was 105.8 (range 84-120) in the adherent group compared with 80.8 (range 40-103) in the non-adherent group. The control group (n = 10), with a mean age of 19.4 years (range 9.7-32.9 years), had a mean FIQ of 102 (range 76-116). Two later-detected patients, aged 18.9 and 18.8 years, had FIQs of 80 and 102, respectively, and two untreated patients, aged 22.4 and 11.7 years, had FIQs of 52 and 53, respectively. With the exception of a significantly higher FIQ in affected siblings (p = 0.0397), there were no significant differences between compliant early-treated individuals and their unaffected siblings (controls). Despite the relatively small numbers, these results suggest that early treatment with excellent biochemical control prevents mental retardation.

[0281] Similar results were obtained in a case-control study reporting on neurodevelopmental, educational, and cognitive outcomes in 32 cases of CBSDH and 25 sibling controls in Qatar (see El Bashir et al. JIMD Rep 2015;21:89-95, incorporated herein by reference in its entirety). The mean age of subjects in this study was 11.2 years (range 0.6-29), and 56% were male. Compared with unaffected siblings, affected individuals had lower overall IQs (particularly in short-term memory, quantitative reasoning, and visuospatial domains), and a significant number of adolescent and adult cases had medical comorbidities and behavioral and emotional problems. Of these, 9 cases of CBSDH (28%) were diagnosed by NBS and treated at 1 month of age; the remainder were diagnosed between 14 and 240 months of age. Presumably due to better dietary and medication adherence early in life, procedural tHcy and Met levels were significantly better in those diagnosed through NBS than in those diagnosed clinically. Significant differences in IQ were observed between early-diagnosed and clinically diagnosed patients. Differences in language domains, attendance at special schools, and access to extra classroom support were not statistically significant between groups, although the "clinically detected" group reported significantly more difficulties.

[0282] Although the number of patients studied here is small, notable differences are seen between children diagnosed at birth and those diagnosed as toddlers. A mean tHcy level of 115 μmol / L in the clinically diagnosed group was associated with poor clinical outcomes and very low IQ.

[0283] Further evidence of the benefit of Hcy-lowering treatment on psychopathology in patients with CBSDH comes from a retrospective chart review of data from all HCU patients presenting at Boston Children's Hospital since 1963 (unpublished data courtesy of M. Almuqbil et al.). Overall, 19 HCU patients were identified; three of these were excluded from the analysis due to the likely confounding presence of methylmalonic acidemia (also associated with psychological deficits) in addition to CBSDH. Of the remaining 16 patients, seven (six with CBSDH and one with cobalamin (Cbl) deficiency) adhered well to early treatment (four with diet alone, two with diet plus betaine, and one with Cbl). Six of these patients had no apparent psychiatric symptoms other than mild cognitive deficits. In contrast, nine patients (seven with pyridoxine-unresponsive CBSDH and two with CblG deficiency) had poor or variable adherence to treatment (two with betaine and diet, one with diet only, three with B vitamins, and two with folic acid and betaine). All seven patients with CBSDH and poor adherence had psychiatric problems, including depression (n = 4), delusional experiences (n = 2), paranoia and paranoid psychosis (n = 1), anxiety and mood dysregulation (n = 1), and ADHD (n = 1), which improved with better metabolic control. Two CblG cases were significantly anxious or agitated. Age, sex, and cognitive level did not appear to significantly differentiate between psychiatrically affected and unaffected individuals. These results suggest that better metabolic control (reduced Hcy and / or Met) has the potential to delay and possibly prevent the development of psychiatric and behavioral conditions in CBSDH patients. However, this study did not confirm whether poorly controlled CBSDH leads to psychopathology or whether comorbidity with psychopathology itself prevents good adherence to treatment outcomes.

[0284] The association between elevated Hcy levels and CNS symptoms, including mental retardation, neurodegenerative diseases, seizures, dystonia, psychosis, cognitive impairment, dementia, and depression, has been well documented in individuals with and without CBSDH (12; Abbott et al. Am J Med Genet 1987;26:959-969; Mudd et al. Disorders of transsulfuration. Scriver CL, Beaudet AL, Sly In Sly WS, Valle D (eds.), The Metabolic and Molecular Basis of Inherited Diseases, 7th ed. New York: McGraw Hill; 2001; 1279-1327; Hidalgo et al., Eur Child Adolesc Psychiatry 2014; 23:235-238; Schimke et al., JAMA 1965; 193:711-719; Smith et al., PLoS One 2010; 5:e12244; Seshadri et al., N Engl J Med 2002; 346:476-483; Bottiglieri et al., J Neurol Neurosurg Psychiatry 2000;69:228-232; Bjelland et al. Arch Gen Psychiatry 2003;60:618-626; Tolmunen et al. Am J Clin Nutr 2004;80:1574-1578; Kaeser et al. J Neurol Neurosurg Psychiatry 1969;32:88-93; Golimbet et al. Psychiatry Res 2009;170:168-171; Clarke et al. Arch Neurol 1998;55:1449-1455; Sinclair et al. Mov Disord 2006;21:1780-1782; Permoda-Osip et al. Neuropsychobiology 2014;69:107-111; Oliveira et al. BMJ Case Rep 2016;2016; Troen et al. Proc Natl Acad Sci USA 2008;105:12474-12479; Sudduth et al. J Cereb Blood Flow Metab 2013;33:708-715; Hainsworth et al. Biochim Biophys Acta 2016;1862:1008-1017; Herrmann et al. Clin Chem Lab Med 2011;49:435-441; Kim et al. J Nutr 2007;137:2093-2097; Selhub et al. Am J Clin Nutr 2000;71:614S-620S; McCaddon et al. al.) Dement Geriatr Cogn Disord 2001;12:309-313; Smallwood et al. Neuropathol Appl Neurobiol 2012;38:337-343; Beydoun et al. BMC Public Health 2014;14:643; Gortz et al. J Neurol Sci 2004;218:109-114; Health Quality O. Vitamin B12 and cognitive function: an evidence-based analysis. Ont. Health Technol. Assess. Ser. 13(23), 1e45. 2013. Ref Type: Online Source; Salagre et al. et al. Eur Psychiatry 2017;43:81-91). The mechanism leading to CNS damage in individuals with elevated Hcy levels is tHcy-mediated neuronal injury (Mudd et al. Disorders of transsulfuration. Scriver CL, Beaudet AL, each of which is incorporated herein by reference in its entirety). AL), Sly WS, Valle D (eds.) The Metabolic and Molecular Basis of Inherited Diseases. 7th ed. New York: McGraw Hill; 2001; 1279-1327; Hainsworth et al. Biochim Biophys Acta 2016; 1862:1008-1017; Stefanello et al. Metab Brain Dis 2007; 22:172-182; Toborek et al. Atherosclerosis 1995; 115:217-224), vascular endothelial damage caused by Hcy-mediated oxidative stress (see Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004;287:R39-R46), neuronal loss (Yeganeh et al. J Mol Neurosci 2013;50:551-557; Heider et al. J Neural Transm Suppl 2004;1-13, both of which are incorporated herein by reference in their entireties), and diminished neural network activity (Gortz et al. J Neurol Sci 2004;218:109-114, both of which are incorporated herein by reference in their entireties). Depression and seizures are thought to be caused, at least in part, by Hcy-mediated decreases in brain adenosine levels and subsequent decreases in norepinephrine and dopamine levels (see Mech et al. J Clin Psychiatry 2016;77:668-671; Domagala et al. Thromb Res 1997;87:411-416; Vivitsky et al. Am J Physiol Regul IntegrComp Physiol 2004;287:R39-R46; Folstein et al. Am J Psychiatry 2007;164:861-867, each of which is incorporated herein by reference in its entirety).

[0285] Animal models of CBSDH (see Lee et al. J Nutr 2005;135:544-548, which are incorporated herein by reference in their entireties) and patients with CBSDH (see El Bashir et al. JIMD Rep 2015;21:89-95; Yap et al. J Inherit Metab Dis 2001;24:437-447; Mech et al. J Clin Psychiatry 2016;77:668-671; Grobe H. Eur J Pediatr, each of which is incorporated herein by reference in its entirety) have been reported. Numerous studies, both in the US and Europe (see, for example, Yap et al., 1980;135:199-203), have shown that early reduction of Hcy levels, induced by a low-Met diet, folic acid / B vitamin supplementation, and / or pyridoxine / betaine therapy, can prevent and sometimes reverse the progression of various neurological disorders. Further evidence is provided by a series of six case studies in patients with CBSDH in which significant reduction, or even normalization, of Hcy levels resulted in complete or partial correction of CNS outcomes (Yap et al., 1980;135:199-203, each of which is incorporated herein by reference in its entirety). et al.) J Inherit Metab Dis 2001;24:437-447; Brenton et al. (J Child Neurol) 2014;29:88-92; Rezazadeh et al. Child Neurol Open 2014;1:2329048X14545870; Kaeser et al. J Neurol Neurosurg Psychiatry 1969;32:88-93; Colafrancesco et al. Eur J Pediatr 2015;174:1263-1266; Yokoi et al. Pediatr Int 2008;50:694-695; Li et al. Pathology 1999;31:221-224).

[0286] These findings demonstrate a strong correlation between Hcy levels and increased risk of CNS damage in patients with CBSDH and the general population. Early Hcy-lowering therapy is essential for normal development in children with early-onset CBSDH and for correction or amelioration of CNS damage in patients diagnosed with CBSDH later in life.

[0287] IX.Definitions As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0288] When a range of values ​​is provided, each intervening value between the upper and lower limits of the range, and any other stated or intervening value in the stated range, is intended to be encompassed within and specifically disclosed within the present disclosure. For example, if a range of 1 μm to 8 μm is stated, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm, as well as ranges of values ​​above 1 μm and below 8 μm, are also intended to be expressly disclosed.

[0289] As used herein, "co-administered" or "co-administration" refers to the administration of two or more therapeutic components that comprise a pharmaceutical composition. As used herein, "pharmaceutical product" refers to a dosage form of a pharmaceutical composition that includes a drug substance of a PEGylated human truncated CBS protein having the amino acid sequence of SEQ ID NO:1 (e.g., 20NHS PEG-CBS).

[0290] As used herein, "drug substance" refers to a PEGylated CBS protein (e.g., 20NHS PEG-CBS) having the amino acid sequence of SEQ ID NO:1. As used herein, a "negative clinical outcome" refers to an undesirable phenotypic outcome that results from a disease, disorder, or condition.

[0291] As used herein, "recombinant," e.g., when used in reference to a cell, nucleic acid, polypeptide, expression cassette, or vector, refers to material that has been modified by the introduction of new portions or alteration of existing portions, or that is identical to, but is produced from or derived from synthetic material, or material that corresponds to the natural or naturally occurring form of the material. For example, recombinant cells express genes (i.e., "exogenous nucleic acids") that are not found within the native (non-recombinant) form of the cell, or express native genes that are expressed at different levels, typically low or not expressed at all.

[0292] Recombinant techniques can include, for example, the use of recombinant nucleic acids, such as cDNAs or antisense sequences encoding proteins, for insertion into an expression system such as an expression vector; the resulting construct is introduced into cells, which express the nucleic acid and, if appropriate, the protein. Recombinant techniques also encompass the ligation of nucleic acids and coding or promoter sequences from different sources into one expression cassette or vector for expression of fusion proteins, constitutive expression of proteins, or inducible expression of proteins.

[0293] As used herein, the terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, humans.

[0294] As used herein, "associated" refers to a correspondence with the onset or manifestation of a disease, condition, or phenotype. Association can be, but is not limited to, by genes that perform housekeeping functions, alterations of which can provide the basis for various diseases and conditions, those that are part of pathways involved in a particular disease, condition, or phenotype, and those that indirectly contribute to the manifestation of a disease, condition, or phenotype.

[0295] As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient that may optionally be included in the compositions of the present disclosure and that does not cause significant adverse toxicological effects to the patient. In particular, in this example, this refers to an excipient that allows the active compound (here, PEGylated htCBS or "20NHS") to be administered without significant adverse toxicological effects to the subject. PEG-CBS”) refers to an excipient that can be placed in the body of a mammalian subject.

[0296] As used herein, the terms "adjuvant," "diluent," or "carrier" refer to any substance, not itself a therapeutic agent, that is used as a carrier for delivering a therapeutic agent and is added to a pharmaceutical composition to make it suitable for administration to a subject, e.g., a mammal, or to improve its handling or storage characteristics, or to enable or facilitate the formation of dosage units of the composition into discrete articles, such as capsules or tablets, suitable for oral administration. The terms "adjuvant," "diluent," or "carrier," as that term is used herein, encompass "excipients," including "pharmaceutically acceptable excipients," "vehicles," "solvents," and the like. Excipients and vehicles include any such materials known in the art that are non-toxic and do not adversely interact with other components of the composition, e.g., any liquid, gel, solvent, liquid diluent, stabilizer, and the like. Administration can refer to oral administration, inhalation, enteral administration, feeding, or inoculation by intravenous injection. Excipients may include standard pharmaceutical excipients and may also include any component that may be used to prepare food and beverages, feed or feed formulations or other foodstuffs for human and / or animal consumption.

[0297] As used herein, "drug" or "pharmacologically active agent" or any other similar term means any chemical or biological material or compound, including peptides, suitable for administration by methods previously known in the art and / or taught in this disclosure to induce a desired biological or pharmacological effect, which may include, but is not limited to, (1) having a prophylactic effect on an organism, preventing an undesired biological effect, such as preventing infection; (2) alleviating a condition caused by a disease, e.g., alleviating pain or inflammation caused as a result of the disease; and / or (3) alleviating, reducing, or completely eliminating a disease from an organism. The effect may be local, e.g., providing a local anesthetic effect, or systemic. This disclosure does not depict novel permeants or novel classes of active agents. Rather, this disclosure is limited to delivery modes of agents or permeants that exist in the prior art or that may later be established as active agents and are suitable for delivery by the present disclosure.

[0298] As used herein, the term "about," particularly in reference to a given amount, is meant to encompass a deviation of + or -5%. As used herein, the term "pharmacologically effective amount" or "therapeutically effective amount," in reference to the present compositions, refers to a non-toxic but sufficient amount of active agent (or composition containing active agent) to provide desired levels in the bloodstream or at the site of action (e.g., intracellularly) in the treated subject, and / or to provide a desired physiological, biophysical, biochemical, pharmacological, or therapeutic response, such as amelioration of homocystinuria manifestations. The exact amount required will vary from subject to subject and will depend on numerous factors, including the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, the intended patient use (i.e., the number of doses administered per day), and patient considerations such as the species, age, and general condition of the subject, the severity of the condition being treated, any additional medications taken by the subject, and the mode of administration. These factors and considerations can be readily determined by one of ordinary skill in the art based on the information provided herein. The appropriate "effective" amount for any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0299] As used herein, the term "nucleic acid" can be in the form of RNA or in the form of DNA, and can include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. The DNA can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (antisense, complementary) strand.

[0300] As used herein, a "mutant" is a mutant protein that has been designed or engineered to have altered properties or functions related to glycosylation, protein stabilization and / or ligand binding.

[0301] As used herein, the terms "native" or "wild-type" with respect to a given cell, polypeptide, nucleic acid, trait, or phenotype refer to the form that is typically found in nature. As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" have their conventional meanings and are used interchangeably to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipid-modification, myristylation, ubiquitination, etc.). Furthermore, the polypeptides described herein are not limited to a particular length. D- and L-amino acids, as well as mixtures of D- and L-amino acids, are included in this definition. The term also does not refer to or exclude post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications (both naturally occurring and non-naturally occurring) known in the art. A polypeptide may be an entire protein or a subsequence thereof. A polypeptide may also refer to an epitope, i.e., an amino acid sequence comprising an antigenic determinant that is substantially responsible for the immunogenic properties of the polypeptide and is capable of eliciting an immune response.

[0302] As used herein, a "position corresponding to" or the like refers to a position of interest (i.e., base number or residue number) in a nucleic acid molecule or protein relative to a position in another reference nucleic acid molecule or protein. Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, e.g., so that the identity between the sequences is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%. The position of interest then receives a number assigned in the reference nucleic acid molecule. For example, if a particular polymorphism in gene X occurs at nucleotide 2073 of SEQ ID NO: X, then to identify the corresponding nucleotide in another allele or isolate, the sequence is assigned and the position flanking 2073 is then identified. Because various alleles may be of different lengths, the position designated 2073 may not be nucleotide 2073, but instead is a position "corresponding" to a position in the reference sequence.

[0303] As used herein, the term "long-term administration" refers to administration of a CBS enzyme, htCBS, or htCBS variant (e.g., having a C15S mutation) conjugated to a PEG moiety for a period of six weeks or longer.

[0304] As used herein, the term "continuous administration" refers to repeated administration of a CBS enzyme, htCBS, or htCBS variant (e.g., having a C15S mutation) conjugated to a PEG moiety throughout the course of the study via subcutaneous injection or implantable osmotic pump.

[0305] Described herein are methods of treating homocystinuria through enzyme therapy (ET) using a pharmaceutical agent described herein, including a PEGylated human truncated CBS protein having a mutation at amino acid position 15 from cysteine ​​to serine.

[0306] Details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described herein. Other features, objects, and advantages of the present disclosure will become apparent from the description. In the description, the singular includes the plural unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present description will control.

[0307] The present disclosure is further illustrated by the following non-limiting examples. [Example]

[0308] Example 1. Experimental Protocol A. Fermentation An expression vector carrying the truncated human CBS coding sequence was transformed into B1-21(DE3) Escherichia coli (E. coli) bacteria, and kanamycin-resistant clones were grown overnight at 37°C in 5 ml of Luria-Bertani (LB) medium containing 30 μg / mL kanamycin on a rotary shaker at 275 rpm. One ml of the overnight culture was added to 100 ml of Terrific Broth (TB) medium containing 30 μg / mL kanamycin and grown overnight. Ten ml of the preculture was then added to 1 L of TB medium containing 0.001% thiamine-HCl pH 8.0, 0.0025% pyridoxine-HCl pH 8.0, 0.3 mM δ-ALA pH 8.0, 150 μM ferric chloride, and 30 μg / mL kanamycin. The culture was then grown at 30°C on a rotary shaker at 275 rpm until the OD600 reached approximately 0.6-0.7, and protein expression was induced by the addition of 1 mM IPTG. Fermentation was continued for an additional 16 hours. Cells were harvested by centrifugation at 6,000 relative centrifugal force (rcf) for 10 minutes at 4°C, washed with ice-cold 0.9% NaCl, recentrifuged as above, and frozen at -80°C. Lysis buffer (20 mM NaH2PO4, pH 7.2, 40 mM NaCl, 0.1 mM PLP) was then added to the cell pellet, which was homogenized in a Dounce homogenizer, treated with lysozyme (2 mg / mL final), incubated on a rocking platform for 1 hour at 4°C, sonicated to reduce viscosity, and centrifuged at 53,000 rcf. The supernatant, containing the soluble fraction, was then stored at -80°C.

[0309] An optimization and scale-up plan was designed to establish the fermentation process for high-level expression of CBS, the amount and addition profile of medium supplements such as 5-aminolevulinic acid (ALA), thiamine (vitamin B1), pyridoxine HCl, and / or ferric chloride, C sources, and their initiation and feeding conditions, temperature, pH, and induction conditions (inducer concentration and length of induction period).

[0310] The highest cell density and best expression level were achieved in minimal salts medium (C source: glycerol) supplemented with a total of 0.05 g / L B6 and 0.5 g / L ALA during a 24-h induction period at 30°C.

[0311] At the end of fermentation (EOF), a cell density at 600 nm (OD600) of approximately 110 ± 10 was achieved, corresponding to a wet weight of approximately 90 ± 10 g / L. The CBS titer of the fermentation process was approximately 2.5-3.0 g CBS / L, as determined by radioactive and colorimetric activity assays.

[0312] B. Purification Assuming that at least three chromatography steps are required, the overall enzyme activity recovery target for the enzyme purified from the cell lysate was 30%. The developed process was evaluated at the scale of a 60 mL capture column.

[0313] 1. Capture process Anion exchange chromatography (AEX) was used as the first capture step in the production of recombinant CBS. The eluate was then refined for further purification by capture onto DEAE-Sepharose resin at near-neutral pH, followed by a linear gradient of KH2PO4. DEAE-Sepharose is a weak exchanger, meaning that the charge of the system was dependent on the ambient pH. Therefore, pH has a close relationship with the dynamic binding capacity (DBC) of the resin. Lowering the binding pH to 6.4 did not decrease the performance of the capture step. When dilution of the cell paste was performed using 5 volumes of disruption buffer for solubilization, the loading time was approximately 30 minutes.

[0314] Washing was performed for 10 column volumes at a near-maximum loading capacity of 7 g / L CBS (equivalent to a DBC of approximately 35 g total protein per L of resin). Elution was performed in the presence of 120 mM NaCl under elution conductivity conditions of 16 mS / cm.

[0315] As long as the cell extract was loaded within a narrow time window, recovery was observed to be approximately 90%. Depending on the CBS titer in the cell extract, this capture step resulted in a purification factor of approximately 3.5.

[0316] 2. Immobilized Metal Affinity Chromatography (IMAC) Using zinc (Zn) ions, Co 2+ IMAC, also known as immobilized metal affinity chromatography, uses binding selectivity for orthogonal separation between CBSs derived from host cell proteins (HCPs). Purification factors of approximately 2.5 have been observed.

[0317] The elution step included 11 mM imidazole in combination with desalting of the CBS fraction. The sodium chloride concentration was reduced from 400 mM (loading / wash) to 50 mM in the elution buffer. Small-scale screening showed that the conductivity significantly affected the elution behavior itself. For example, it was possible to elute CBS with pure water alone, without imidazole. Increasing the amount of sodium chloride in the elution buffer increased the stability of CBS. At 150 mM sodium chloride, the elution behavior only slightly changed.

[0318] Using HEPES as the buffer system, DBC concentrations of 12–15 g / L could be measured. Furthermore, sample stability was increased with respect to precipitation, as well as degradation, which was reduced. Degradation was prevented by the addition of EDTA using a HEPES buffer, as opposed to the phosphate buffer system used here. In scale-up, EDTA and ammonium sulfate, which inhibit degradation, were added immediately after elution was completed. Furthermore, to keep incubation times short in this situation, the following polishing was performed as quickly as possible. The IMAC elution pool was loaded onto a hydrophobic interaction chromatography (HIC) column within 3 h of polishing.

[0319] 3.Refinement process The final chromatographic step in the CBS purification column was based on hydrophobic interactions. The main column separated the target protein, CBS, from the majority of HCPs by varying elution conditions. The majority of HCPs bound more strongly to HIC than CBS, indicating that CBS is less hydrophobic than the majority of the remaining HCPs.

[0320] In contrast to IMAC, HIC has low binding separation (less than 10%). More than 90% of the remaining HCP impurities were separated from HIC by the elution step. CBS recovery in HIC was approximately 95%. DBC was between approximately 16 g / L and approximately 18 g / L. The performance of this polishing step was extremely robust. The pH of the elution step was between 6 and 8. The buffer system (phosphate or 20 mM HEPES) had no measurable effect.

[0321] The purification factor for this polishing step was approximately 1.1 due to the low residual HCP content in the load, typical of the final chromatography step. The purity of the final CBS among all performed downstream purification processes was similarly high, indicating that this polishing step compensated for various degrees of impurities in the load.

[0322] Adaptations can be performed to fit process steps into existing equipment and allow for later scale-up to 100 L fermentation volumes for production. To ensure reliable product quality, EDTA (e.g., 10 mM) can be added to some process steps to prevent product degradation caused by metalloproteases.

[0323] C. PEGylation The PEGylation reaction behaved like a second-order reaction due to product-induced inhibition. Hydrolysis of NHS-PEG could not be the reason for the inhibition of the reaction, and the final PEGylation pattern was reached after 4 h of PEGylation, so it was observed that the remaining (approximately 50%) NHS-PEG was still active. Interestingly, the addition of further NHS-PEG to the reaction mix increased the PEGylation pattern. In the absence of DMSO, the CBS concentration was 8 g / L in the reaction mixture to obtain the desired PEGylation pattern without increasing the NHS-PEG to CBS ratio.

[0324] D. Data and Statistical Analysis Table 4 provides details of the software, analytical tools and algorithms used for data analysis in the Examples herein.

[0325] [Table 4]

[0326] All chromatograms were reviewed by an analyst to ensure satisfactory chromatographic peak shapes and peak integration. Manually transferred data were cross-checked against source data that was part of the test raw data. Run acceptance criteria were established prior to analysis based on results from calibration standards, QCs, and blank QCs. Data are presented as mean ± standard error of the mean (SEM).

[0327] Statistical analysis was performed using ANOVA followed by Tukey's multiple comparison test to determine significance. E. CBS Activity Assay Based on Conversion of Radiolabeled Ser CBS catalyzes the β-substitution reaction in which serine (Ser) condenses with Hcy to form Cth in a pyridoxal 5'-phosphate (PLP)-dependent manner. The activity of the CBS enzyme was determined by a radioisotope assay using 14C-labeled Ser as a substrate: 14 CL-Ser+L-Hcy→ 14 CL-Cth+H2O A 7 μl (420 ng total) aliquot of pure 20NHS PEG-CBS or 7 μl of plasma (no dilution necessary for D25 and D27 samples) in dilution buffer (0.1 M Tris-HCl pH 8.6 containing 1 mM DTT, 10 μM PLP, and 0.5 mg / ml bovine serum albumin (BSA)) was added to 88 μl of reaction mixture (10 mM L-Ser, 0.5 mM PLP, 0.5 mg / ml BSA, and 0.3 μCi The reaction mixture was added to 0.1 M Tris-HCl (pH 8.6) containing L-[C(U)]-Ser. The sample was equilibrated to 37°C by incubating in a water bath for 5 min. The reaction was initiated with 5 μl of 0.2 M Hcy (final concentration 10 mM) and incubated at 37°C for an additional 30 min.

[0328] Assay initiation and sampling of the resulting mixture were staggered so that each reaction lasted exactly 30 minutes. The radioactive product (Cth) was separated from the labeled substrate (Ser) using descending-flow paper chromatography. To terminate the reaction, the assay mixture was cooled in an ice bath, and a 20 μl aliquot was pipetted onto grade 3 CHR chromatography paper (Whatman, GE Healthcare, Pittsburgh, PA, USA) for separation. The 14C-Cth formed in the reaction was separated from 14C-Ser by overnight elution with 2-propanol:formic acid:HO (75:5.7:18.9 v / v). Standards containing a mixture of Cth and Ser were run alongside the sample on each side of the chromatography paper. The chromatography paper was dried, and the standard lane was stained with acidic ninhydrin solution. A section of each sample lane containing labeled Cth was excised, submerged in 5 ml of Opti-flour scintillation cocktail (PerkinElmer, North Billerica, MA, USA), and counted in a Beckman LS-3801 scintillation counter. A sample without enzyme was used as a blank to monitor background radioactivity, which was subtracted from each sample count. For pure enzyme controls, CBS specific activity values ​​were expressed as enzyme units (the amount of enzyme that produces 1 μmol of Cth / h) per mg of CBS (i.e., U / mg protein). For plasma samples, activity values ​​were expressed as milliunits (the amount of enzyme that produces 1 nmol of Cth / h) per μl of plasma (i.e., mU / μl plasma).

[0329] These stock solutions, with a nominal range of 25 mg / ml (24.8-26.7 mg / ml in phosphate-buffered saline, pH 7.4 (PBS)), were stored in aliquots at -80 °C. On each treatment day, single-use fresh solutions of the enzyme were prepared by diluting the stock solution in PBS to a final concentration of 1 mg / ml, and these were delivered to mice at a dose of 7.5 mg / kg. The volume injected to administer the target dose was calculated based on the body weight on the indicated day. Any remaining solution was discarded upon completion of the day's injections.

[0330] F. Western Blotting The pharmaceutical agents were injected into four I278T- / - mice, and blood was collected 24 hours after the first injection and 72 hours after the last injection to serve as controls. D25 and D27 plasma samples from all groups in Example 5 were analyzed by Western blotting to detect potential in vivo dePEGing. Plasma samples (4 μl per lane) were loaded onto a gradient (4%-20%) MiniProtean TGX gel (Bio-Rad, Hercules, CA, USA), and proteins were separated by electrophoresis under denaturing, reducing conditions. Molecular weight markers (Precision Plus Protein Dual Color Standard, Bio-Rad) and aliquots from each preparation (150-500 ng per lane) were similarly treated and electrophoresed alongside the plasma samples. After electrophoretic separation, protein bands were transferred to a PVDF membrane (Bio-Rad). Individual membranes were blocked overnight at 4°C in blocking solution (5% nonfat milk in PBS containing 0.02% Tween 20). The membranes were then washed and incubated for 1 hour with rabbit polyclonal anti-hCBS antibody (Orphan Technologies, UCD Kraus Laboratory, batch number R2B2, antiserum diluted 1:5000 in blocking solution). The membranes were then washed and incubated for 30 minutes with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Jackson Laboratories, Bar Harbor, ME, USA, diluted 1:5000 in blocking solution). After washing the membrane, bands were incubated with a chemiluminescent substrate (SuperSignal WestPico, ThermoFisher Scientific, Waltham, MA, USA) for 5 min and subsequently visualized by signal capture on Clearblue X-ray film (CL-Xposure Film, ThermoFisher Scientific).The developed films were scanned using a flatbed scanner (Perfection V550 Photo Color Scanner, Epson, Long Beach, CA, USA).

[0331] G. Chemicals Unless otherwise stated, all materials were purchased from Sigma or Fisher Scientific. L-[U-14C]-Serine was obtained from Perkin Elmer Life Sciences.

[0332] H. Plasma Collection and Analysis A single-use lancet for submandibular bleeding was used to collect blood into BD Microtainer PST tubes (Becton, Dickinson and Company, New Jersey, USA) containing lithium heparin. The tubes were then centrifuged at 10,000 × g for 5 minutes, and the plasma was subsequently transferred to 1.5 ml tubes and stored at -80°C. Plasma sulfur amino acid metabolites were determined by stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS / MS) as described elsewhere.

[0333] I. Animal Testing Studies were performed using a CBS knockout strain of mice expressing the human I278T mutant CBS transgene (I278T CBS- / - (I278T- / -) mice). A pair of heterozygous transgenic I278T mice on a C57BL6 background was provided by Dr. Warren Kruger (Fox Chase Cancer Center, Philadelphia, PA, USA). The mice had the mouse CBS gene knocked down and express I278T human CBS under the control of the metallothionein promoter (see Wang, et al. (2005) Hum Mol Genet 14, 2201-2208, incorporated herein by reference in its entirety). The mutant enzyme has approximately 2%-3% of the CBS WT activity, and as a result, transgene expression rescues the neonatal lethality typically observed in CBS knockout homozygous mice (see Watanabe et al. (1995) Proceedings of the National Academy of Sciences USA 92, 1585-1589; Maclean et al. (2010) Molecular Genetics and Metabolism 101, 163-171, each of which is incorporated herein by reference in its entirety). Unless otherwise indicated, animals were maintained on standard irradiated extruded rodent chow (Teklad 2920X, Envigo, Indianapolis, IN, USA).

[0334] To generate CBS homozygous transgenic test animals, homozygous or heterozygous males were mated with heterozygous females to generate litters containing CBS homozygous (- / -) pups, among other genotypes, all expressing the I278T transgene. Zinc (25 mM zinc sulfate in drinking water) was provided to mated and newborn mice to induce transgene expression. The homozygous strain (I278T- / -) has a homocystinuria (HCU) phenotype and serves as a model for CBSDH, but other resulting genotypes were not used in this study (see Wang et al. (2005) Hum Mol Genet 14, 2201-2208, incorporated herein by reference in its entirety).

[0335] All animal procedures used in this study were reviewed and approved by the University of Colorado Denver IACUC, which complies with all federal, state, and local laws, regulations, and policies. The University of Colorado Denver is an AAALAC-accredited (number 00235), Public Health Service-insured (number A 3269-01), and USDA-accredited (number 84-R-0059) institution. Procedures involving mice were performed under IACUC-approved protocol number B-49414(03)1E.

[0336] Mice were bred and genotyped in our facility as previously described in Wang et al. (2005) Hum Mol Genet 14:2201-2208, which is incorporated herein by reference in its entirety. Pairs were maintained on extruded standard diet 2920X (Envigo, CA, USA) and water containing 25 mM ZnCl to induce transgene expression and thus rescue homozygous I278T pups from neonatal mortality. After weaning at 21 days of age, homozygous I278T mice and their WT siblings were assigned to one of eight groups. At 24 days of age, mice were switched to a normal (Envigo TD.170063, 4% Met; groups A, B, C, and D) or restricted methionine content (Envigo TD.110591, 0.5% Met; groups E, F, G, and H) amino acid diet. In addition to the new diet on day 24, mice also received subcutaneous injections of PBS vehicle (groups A, C, E, and G) or 10 mg / kg 20NHS PEG-CBS (groups B, D, F, and H) three times a week (Monday, Wednesday, and Friday) until 22 weeks of age. Mice were weighed weekly, and weights were used to calculate the weekly 20NHS PEG-CBS injection dose.

[0337] J. Enzyme Storage and Preparation The liquid formulation of 20NHS PEG-CBS was used in the stability studies performed under cGMP described herein, which yielded the following samples: a control enzyme or "control preparation" stored below -65°C (T0-80C), and enzyme samples incubated under accelerated stability conditions at 25°C for 2 days (T2D-25C), 1 month (T1M-25C), and 6 months (T6M-25C).

[0338] Example 2. Determination of the reduction in the level of PEGylation in pharmaceutical preparations Drug product preparations with varying degrees of dePEGylation were generated for accelerated stability testing conducted under Good Manufacturing Practice (GMP) conditions for drug products by incubating the drug products at 25° C. for 2 days, 1 month, or 6 months. Control samples were stored within the recommended temperature range, i.e., below −65° C.

[0339] Differences in PEGylation between preparations were confirmed by reverse-phase high-performance liquid chromatography (RP-HPLC). Ten highly PEGylated and less PEGylated species were distinguished, as well as CBS, which had no PEGylation and in which the heme cofactor (P10) was released upon enzyme denaturation. The degree of dePEGylation in each sample was determined by comparing the relative areas of the peaks corresponding to the variably PEGylated or fully dePEGylated species. The RP-HPLC results are shown in Table 5. "CBS" represents the native (unmodified) enzyme, and the remaining peaks represent preparations with various levels of PEGylation.

[0340] [Table 5]

[0341] RP-HPLC analysis indicated significant dePEGylation in the preparations incubated at 25°C for 1 month, and even 6 months, compared with the control samples. As shown in Table 5, these samples exhibited a higher proportion of peaks corresponding to less PEGylated forms (e.g., P3) and a concomitant lower abundance of peaks corresponding to more PEGylated forms (e.g., P6 and P7). In addition, the peak corresponding to unmodified (or fully dePEGylated) CBS was most prominent in the 6-month-incubated preparation. However, in the preparation incubated at 25°C for 2 days, the distribution of variable PEGylated species was more similar to that of the control enzyme, which was always stored at -65°C or below.

[0342] Example 3. Efficacy of preparations with reduced PEGylation levels in mice To assess the bioequivalence between the drug and its dePEGylated preparation, efficacy was analyzed in I278T CBS- / - (I278T- / -) mice, a model that recapitulates the biochemical sequelae of CBSDH, i.e., abnormal plasma levels of methionine (Met) metabolites, including elevated homocysteine ​​(Hcy) and depressed cysteine ​​(Cys). Plasma aminothiols (total Cys and Hcy) and remaining amino acids, as well as SAM and SAH, were measured by LC-MS / MS performed as described in Arning et al. (2016) Methods Mol Biol 1378, 255-262, which is incorporated herein by reference in its entirety.

[0343] I278T CBS- / - (I278T- / -) mice are defective in the mouse CBS gene and express the I278T mutant human CBS gene, which carries the most prevalent pathogenic mutation in CBSDH patients. These mice express approximately 2%-3% wild-type CBS activity and have a homocystinuria (HCU) phenotype (Wang, et al. (2005) Hum Mol Genet 102:101-102, incorporated herein by reference in its entirety). 14, 2201-2208). This includes elevated plasma and tissue levels of Hcy, Met, and S-adenosylhomocysteine ​​(SAH), and a concomitant decrease in plasma Cth and Cys levels. This mouse strain was originally developed as the Fox Chase Warren Cancer Center, Philadelphia, PA, USA Obtained from Professor Warren D. Kruger.

[0344] Efficacy was tested using adult I278T- / - mice, both male and female, maintained on a standard (REG) diet. At the start of the study, mice were divided into four groups corresponding to the four preparations. Blood was collected for baseline measurements at least one week prior to dosing. Blood was collected in plasma preparation tubes containing lithium heparin and gel (Grainer BioOne). Blood samples were collected from the submandibular vein of conscious test animals using disposable lancets designed for submandibular sampling (Bio-One, Monroe, NC, USA, or equivalent from other vendors). Plasma was collected f...

Claims

1. (a) an isolated cystathionine beta synthase (CBS) protein comprising SEQ ID NO:1; and (b) a PEG molecule covalently attached to said CBS protein. A drug substance comprising:

2. 2. The drug substance of claim 1, wherein the PEG molecule is ME-200GS.

3. A pharmaceutical composition comprising the drug substance of claim 1 or 2 and a pharmaceutically acceptable excipient, adjuvant, diluent or carrier.

4. 4. The pharmaceutical composition of claim 3, which is lyophilized.

5. Upon reconstitution, the reconstituted liquid formulation: (a) the drug substance at a concentration of about 20-30 mg / ml; (b) about 15 mM potassium phosphate; and (c) about 8% (w / v) trehalose 5. The lyophilized formulation of claim 4, comprising:

6. The unit dose of the formulation is (a) about 25 mg of the drug substance; and (b) 1 mL of water 6. The reconstituted formulation of claim 5, comprising:

7. 10. A method of treating homocystinuria in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of the pharmaceutical composition of any one of claims 4 to 6.

8. 8. The method of claim 7, wherein the therapeutically effective amount is a dosage selected from the range of about 0.25 mg / kg to about 10 mg / kg.

9. 9. The method of claim 8, wherein the dosage is about 0.33 mg / kg.

10. 9. The method of claim 8, wherein the dosage is about 0.66 mg / kg.

11. 9. The method of claim 8, wherein the dosage is about 1.0 mg / kg.

12. 9. The method of claim 8, wherein the dosage is about 1.5 mg / kg.

13. The method of any one of claims 7 to 12, further comprising administering to the subject at least one selected from the group consisting of pyridoxine, vitamin B6, and betaine.

14. The method of any one of claims 7 to 13, wherein the subject is on a methionine (Met) restricted diet.

15. The method of any one of claims 7 to 14, further comprising administering an antiplatelet drug.

16. 16. The method of claim 15, wherein the antiplatelet agent is a warfarin blood thinner or anticoagulant.

17. 17. The method of any one of claims 7 to 16, wherein the administering step occurs about once every three days.

18. 17. The method of any one of claims 7 to 16, wherein the administering step occurs about once daily.

19. 17. The method of any one of claims 7 to 16, wherein the administering step occurs about once a week.

20. 20. The method of any one of claims 7 to 19, wherein the administering step is repeated for about 6 weeks.

21. 20. The method of any one of claims 7 to 19, wherein the administering step is repeated for about 3 months.

22. 20. The method of any one of claims 7 to 19, wherein the administering step is repeated for about six months.

23. 20. The method of any one of claims 7 to 19, wherein the administering step is repeated for more than six months.

24. 20. The method of any one of claims 7 to 19, wherein the administering step is repeated for the remainder of the subject's life.

25. 10. A method of lowering homocysteine ​​(Hcy) levels in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of the pharmaceutical composition of any one of claims 4 to 6.

26. 26. The method of claim 25, wherein the Hcy level is less than about 80 μM after the administering step.

27. 26. The method of claim 25, wherein the Hcy level is reduced by up to 10% after the administering step.

28. 26. The method of claim 25, wherein the Hcy level is reduced by up to 20% after the administering step.

29. 26. The method of claim 25, wherein the Hcy level is reduced by up to 30% after the administering step.

30. 26. The method of claim 25, wherein the Hcy level is reduced by up to 40% after the administering step.

31. 26. The method of claim 25, wherein the Hcy level is reduced by up to 50% after the administering step.

32. 26. The method of claim 25, wherein the Hcy level is reduced by up to 60% after the administering step.

33. 26. The method of claim 25, wherein the Hcy level is reduced by up to 70% after the administering step.

34. 26. The method of claim 25, wherein the Hcy level is reduced by up to 80% after the administering step.

35. 26. The method of claim 25, wherein the Hcy level is reduced by up to 90% after the administering step.

36. 36. The method of any one of claims 25-35, wherein the Hcy level is in the range of about 10 μM to about 20 μM after the administering step.

37. 36. The method of any one of claims 25-35, wherein the Hcy level is less than 10 μM after the administering step.

38. 36. The method of any one of claims 25-35, wherein the Hcy level is about 55 μM after the administering step.

39. 39. The method of any one of claims 25 to 38, wherein the therapeutically effective amount is a dosage selected from the range of about 0.25 mg / kg to about 10 mg / kg.

40. 40. The method of claim 39, wherein the dosage is about 0.33 mg / kg.

41. 40. The method of claim 39, wherein the dosage is about 0.66 mg / kg.

42. 40. The method of claim 39, wherein the dosage is about 1.0 mg / kg.

43. 40. The method of claim 39, wherein the dosage is about 1.5 mg / kg.

44. 40. The method of claim 39, wherein the dosage is about 7.0 mg / kg.

45. 40. The method of claim 39, wherein the dosage is about 10 mg / kg.

46. 40. The method of claim 39, wherein the dosage is less than 10 mg / kg.

47. 47. The method of any one of claims 25 to 46, further comprising administering to the subject at least one selected from the group consisting of pyridoxine, vitamin B6, and betaine.

48. 48. The method of any one of claims 25 to 47, wherein the subject is on a methionine (Met) restricted diet.

49. 49. The method of any one of claims 25 to 48, further comprising administering an antiplatelet agent.

50. 50. The method of claim 49, wherein the antiplatelet agent is a warfarin blood thinner or anticoagulant.

51. 51. The method of any one of claims 25-50, wherein the administering step occurs about once every three days.

52. 51. The method of any one of claims 25 to 50, wherein the administering step occurs about once daily.

53. 51. The method of any one of claims 25 to 50, wherein the administering step occurs about once a week.

54. 51. The method of any one of claims 25-50, wherein the administering step is repeated for about 6 weeks.

55. 51. The method of any one of claims 25-50, wherein the administering step is repeated for about 3 months.

56. 51. The method of any one of claims 25-50, wherein the administering step is repeated for about six months.

57. 51. The method of any one of claims 25-50, wherein the administering step is repeated for more than six months.

58. 51. The method of any one of claims 25 to 50, wherein the administering step is repeated for the remainder of the subject's life.

59. A method of increasing cysteine ​​(Cys) levels in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of the pharmaceutical composition of any one of claims 4 to 6.

60. A method for increasing cystathionine (Cth) levels in a subject, comprising administering to the subject a therapeutically effective amount of a formulation of the pharmaceutical composition of any one of claims 4 to 6.

61. 10. A method of treating, alleviating or preventing a negative clinical outcome associated with the ocular, skeletal, vascular and / or central nervous system in a subject, comprising administering to said subject a therapeutically effective amount of a formulation of the pharmaceutical composition of any one of claims 4 to 6.

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