Treatment of homocystinuria and hyperhomocysteinemia using cystathionine-gamma-lyase

Abstract

To provide compositions for treating homocystinuria and hyperhomocysteinemia patients.SOLUTION: A pharmaceutical composition for treating a subject having, or at risk of developing, homocystinuria or hyperhomocysteinemia, the composition comprising a modified human cystathionine-γ-lyase (CGL) enzyme, wherein the modified human CGL enzyme comprises at least the following substitutions relative to a specific native human CGL amino acid sequence: isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353, and wherein the pharmaceutical composition is administered at an enzyme dose of 0.05 mg / kg to 1 mg / kg at a frequency of one dose per day to one dose per month.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present disclosure generally relates to methods for treating patients with homocystinuria and hyperhomocysteinemia.

Background Art

[0002] Homocystinuria is an autosomal recessive disorder of methionine metabolism, which results in a wide range of severe consequences with irreversible pathological conditions and reduced average life expectancy due to elevated levels of homocysteine and homocysteine metabolites (such as homocysteine, homocysteine-cysteine complex, etc.) in plasma and urine. Homocystinuria is a genetic defect in the sulfur transfer pathway (homocystinuria I) or the methylation pathway (homocystinuria II and III). In addition to increased plasma levels of homocysteine and homocysteine, affected subjects have elevated plasma levels of methionine and S-adenosylhomocysteine, as well as reduced levels of plasma cystathionine and cysteine. The main clinical features include developmentally delayed learning difficulties and intellectual disabilities, skeletal abnormalities (such as excessive height, long and thin limbs [dolichostenomelia], scoliosis, funnel chest, Marfanoid appearance), ectopia lentis (displacement of the eye lens), glaucoma, and / or severe myopia, premature thromboembolic vascular events, and fatty liver.

[0003] Normally, homocysteine is metabolized to cystathionine via the sulfur transfer pathway, and ultimately homocysteine or cysteine is converted to methionine by the remethylation pathway (Figure 1). Homocystinuria is caused by a mutation in one of the genes related to the sulfur transfer pathway or the remethylation pathway, such as cystathionine beta-synthase (CBS), metabolism of cobalamin (cbl)-related D (MMADHC), 5,10-methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate homocysteine methyltransferase (MTR), or 5-methyltetrahydrofolate homocysteine methyltransferase reductase (MTRR).

[0004] Patients with homocystinuria are unable to properly process certain components of proteins (amino acids). There are multiple forms of homocystinuria, which are distinguished by their signs and symptoms as well as their genetic causes. Homocystinuria due to mutations in the CBS gene, also known as classical homocystinuria (OMIM 236200), is the most common form of homocystinuria. The function of the CBS enzyme is to convert homocysteine to cystathionine (the first step in the sulfur transfer pathway), but a deficiency or defect in CBS results in the accumulation of homocysteine, which can also lead to the accumulation of the amino acid methionine by the remethylation of homocysteine. Individuals with the common form of homocystinuria are characterized by myopia, dislocation of the lens in the front part of the eye, an increased risk of abnormal blood clotting, and brittle bones that are prone to fractures (osteoporosis) or other skeletal abnormalities. Some affected individuals also have problems with developmental delay and learning ability.

[0005] More rarely, homocystinuria can be caused by mutations in the MTHFR, MTR, MTRR, and MMADHC genes. The enzymes encoded by these genes play a role in converting homocysteine to methionine, but the mutations result in the accumulation of homocysteine in the body. Individuals may exhibit intellectual disability, failure to grow and gain weight at the expected rate (failure to thrive), seizures, problems with movement, and a blood disorder known as megaloblastic anemia. Megaloblastic anemia occurs when there are fewer red blood cells (anemia) and the remaining red blood cells are larger than normal (megaloblastic). The signs and symptoms of homocystinuria typically appear within the first year of life, but some mildly affected individuals may not develop features until later in childhood or adulthood.

[0006] As a result of sufficient evidence of homocystinuria due to CBS deficiency, it has been demonstrated that the destructive complications of the disease are strongly associated with chronic exposure to elevated plasma total homocysteine (tHcy) levels. Previous studies have demonstrated that reducing plasma tHcy has a consistent favorable effect in reducing disease-related complications. Therefore, treatment guidelines defined by experts emphasize that the main goal in treating homocystinuria due to CBS deficiency is to lower plasma tHcy.

[0007] Patients with homocystinuria due to CBS deficiency are known to have symptoms related to cognitive, behavioral, and psychiatric disorders that have a profound impact on the patient and their family. Many homocystinuria patients with CBS deficiency have mental and chronic behavioral disorders, some of which may not fit into standard diagnostic categories. Reducing homocysteine levels through disease management is thought to improve behavior and even intellectual ability. Therefore, it is reasonable to hypothesize that the management of adaptive behavior is clinically important in subjects with homocystinuria due to CBS deficiency and that early treatment to lower plasma tHcy may lead to improvement in adaptive behavior.

[0008] In current disease management for subjects with homocystinuria due to CBS deficiency, dietary protein and methionine restriction is carried out with the aim of reducing plasma tHcy concentration to the recommended level while maintaining appropriate nutrition. To evaluate the response to diet, frequent metabolic monitoring by plasma amino acid analysis is required. In addition, the degree of protein restriction in the diet of subjects with homocystinuria due to CBS deficiency is difficult to maintain, and compliance is poor. Furthermore, this degree of dietary protein restriction can give rise to problems regarding long-term malnutrition and social acceptability, which results in poor compliance and an overall disease burden. Therefore, adhering to a strict amino acid-restricted diet is difficult for many subjects with homocystinuria. Therapeutic options that allow patients to freely perform dietary restrictions bring about a major improvement in the quality of life. That is, when patients can biochemically control and maintain low tHcy levels in plasma, they can freely diet while increasing their intake of natural proteins and reducing or eliminating medical foods and amino acid supplements.

[0009] The unmet medical needs of patients with homocystinuria due to CBS deficiency are mainly in the following three aspects. First, the existence of therapy-resistant patients who are receiving optimal standard disease management but cannot consistently maintain tHcy levels below the desired target concentration. Second, the existence of patients with a high risk of non-compliance with a low-methionine diet and / or adjuvant therapy (pyridoxine and / or betaine therapy), and a high risk of severe complications including sudden death (for example, those in their late teens and young adults, or patients with behavioral problems or intellectual disabilities). Third, the existence of patients who cannot tolerate available adjuvant therapies due to either safety issues or other side effects due to having tHcy levels above the desired target concentration.

[0010] Current therapies are severely limited by poor compliance and limited efficacy in controlling plasma tHcy. There is an apparently unmet need for alternative therapies that provide better control of plasma tHcy and allow for dietary freedom and discontinuation of betaine. Such treatments would minimize chronic and lifelong exposure to toxic levels of plasma tHcy, thereby reducing the severe consequences of homocystinuria and the burden on patients and their caregivers. SUMMARY OF THE INVENTION

[0011] One aspect of the invention relates to a method of treating a subject having or at risk of developing homocystinuria or hyperhomocysteinemia. In one or more embodiments of this aspect, the method comprises administering a formulation comprising a modified human cystathionine-gamma-lyase (CGL) enzyme comprising at least the following substitutions relative to the native human CGL amino acid sequence (SEQ ID NO: 1): isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353. In one or more embodiments, the formulation is administered at a frequency of 1 dose per day to 1 dose per month at an enzyme dose of about 0.05 mg / kg to about 4 mg / kg.

[0012] In one or more embodiments, the enzyme is conjugated to one or more PEG units.

[0013] In one or more embodiments, the formulation further comprises a pharmaceutically acceptable carrier.

[0014] In one or more embodiments, the formulation is a 2 mL liquid supplied in a 5 mL vial.

[0015] In one or more embodiments, the formulation comprises an enzyme concentration of about 1 mg / mL to about 50 mg / mL. In one or more embodiments, the formulation comprises an enzyme concentration of about 1 to about 20 mg / mL.

[0016] In one or more embodiments, the formulation is administered intravenously or subcutaneously.

[0017] In one or more embodiments, the formulation is administered at a dose of once per week.

[0018] In one or more embodiments, the formulation is administered intravenously for 4 weeks and then subcutaneously in subsequent weeks.

[0019] In one or more embodiments, the formulation is administered to the subject at a dose of about 0.1 mg / kg to about 1 mg / kg. In one or more embodiments, the formulation is administered once a week at a dose of about 0.1 mg / kg to about 1 mg / kg. In one or more embodiments, the formulation is administered to the subject at a dose of about 0.15 mg / kg, about 0.45 mg / kg, or about 1 mg / kg.

[0020] In one or more embodiments, the formulation is diluted in physiological saline before being administered intravenously to the subject.

[0021] In one or more embodiments, the subject is a human patient.

[0022] In one or more embodiments, the subject has a total plasma homocysteine level of greater than 80 μM before starting therapy by administering the formulation.

[0023] In one or more embodiments, the subject is at least 12 years old.

[0024] In one or more embodiments, the subject is maintained on an individualized diet.

[0025] In one or more embodiments, the subject is maintained on a methionine-restricted diet.

[0026] In one or more embodiments, the method reduces total plasma homocysteine levels. In one or more embodiments, the method reduces total plasma homocysteine levels to about 80 μM or less. In one or more embodiments, the method reduces total plasma homocysteine levels to about 50 μM or less. In one or more embodiments, the method reduces total plasma homocysteine levels to about 15 μM or less.

Brief Description of the Drawings

[0027] Further features of the invention will become apparent from the following written description and the accompanying figures.

[0028]

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Mode for Carrying Out the Invention

[0029] There is an unmet medical need for patients with homocystinuria and hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency. Engineered human cystathionine gamma-lyase (CGL) was designed to degrade homocysteine and homocystine. Modifications to CGL introduced into the CGL active site (e.g., PEGylation and / or polyhistidine tag) and various mutations (e.g., E59I, S63L, L91M, R119D, K268R, T311G, E339V, I353S) resulted in molecules with high substrate specificity for homocysteine and homocystine but not for cystathionine, the natural substrate. In the case of classical homocystinuria patients, the engineered enzyme is expected to provide an alternative route for the enzymatic degradation of high plasma total homocysteine levels (tHcy), thereby ameliorating the adverse effects of CBS enzyme deficiency in the transsulfuration pathway. Current therapies are severely limited by poor compliance and limited efficacy in controlling plasma tHcy. There is an apparently unmet need for alternative therapies that provide better control of plasma tHcy and allow for liberalization of diet and discontinuation of betaine. The engineered enzyme is advantageous for minimizing chronic, lifelong exposure to toxic levels of plasma tHcy, thereby reducing the severe consequences of homocystinuria (e.g., osteoporosis, neurological, and psychological complications, and thromboembolic events), as well as the burden on patients and their caregivers.

[0030] An engineered human cystathionine gamma-lyase (CGL-ILMDRGVS) having the mutations E59I, S63L, L91M, R119D, K268R, T311G, E339V, I353S was developed as a therapeutic agent for homocystinuria and hyperhomocysteinemia. CGL-ILMDRGVS (and its PEGylated form: CGL-ILMDRGVS-PEG) is a pyridoxal phosphate (PLP)-dependent tetramer that catalyzes the conversion of homocysteine and homocystine to alpha-ketobutyrate, thio-homocysteine, hydrogen sulfide, and ammonia. CGL-ILMDRGVS also degrades methionine and cysteine, but with potencies approximately 20- and 10-fold lower than those of its preferred substrates, homocysteine and homocystine. CGL-ILMDRGVS was developed by protein engineering of the human enzyme cystathionine gamma-lyase (CGL, EC 4.4.1.1). PEGylation of CGL-ILMDRGVS yields CGL-ILMDRGVS-PEG compounds with an extended serum half-life. Similar to CGL, CGL-ILMDRGVS-PEG (and CGL-ILMDRGVS) forms homotetramers non-covalently. Covalent binding of the cofactor pyridoxal phosphate (PLP) at the active site of CGL-ILMDRGVS is essential for its catalytic activity. The effects of cystathionine-gamma-lyase (CGL) variant enzymes on pharmacokinetics (PK) and pharmacodynamics (PD), including plasma total homocysteine (tHcy) levels, were determined to find an effective human dose for treating homocystinuria and hyperhomocysteinemia. The biochemical properties of CGL-ILMDRGVS are summarized in Table 1, and the primary amino acid sequence (including the mutated amino acids) is shown in Figure 2. [Table 1]

[0031] Accordingly, various embodiments of the invention relate to the administration of modified CGL enzymes such as CGL-ILMDRGVS and CGL-ILMDRGVS-PEG.

[0032] Definitions As described herein, the terms "enzyme" and "protein" and "polypeptide" refer to compounds containing amino acids linked via peptide bonds and are used interchangeably.

[0033] As used herein, the term "fusion protein" refers to a chimeric protein comprising a protein or protein fragment operably linked in a non-natural manner.

[0034] As described herein, the term "half-life" (1 / 2-life) refers to the time required for the concentration of a polypeptide to decrease by half in vitro (e.g., measured in cell culture medium) or in vivo (e.g., measured in serum), for example, after injection into a mammal. Methods for measuring "half-life" include the use of antibodies specific for CGL or PEG used in an ELISA (enzyme-linked immunosorbent assay) format, whereby the physical amount of the protein is measured as a function of time. Other methods for measuring half-life include determining the catalytic activity of an enzyme agent as a function of time by any assay that detects the production of any substrate resulting from the conversion of homocysteine to products such as alpha-ketobutyrate, methanethiol, and / or ammonia.

[0035] The term "linker" means a compound or moiety that acts as a molecular bridge to operably link two different molecules, with one part of the linker operably linked to a first molecule and another part of the linker operably linked to a second molecule.

[0036] As described herein, the term "PEGylation" refers to a conjugate with polyethylene glycol (PEG), which is widely used as a drug carrier considering its high biocompatibility and ease of modification. PEG can be attached (e.g., covalently) to an active agent by chemical methods via the hydroxy groups at the ends of the PEG chains. However, PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids retain the biocompatibility properties of PEG but have the added advantage of multiple attachment points per molecule (thereby resulting in a higher drug loading capacity) and are being investigated as novel biomaterials that can be synthetically designed to fit various applications.

[0037] As described herein, the term "gene" refers to a DNA sequence that includes the control and coding sequences necessary for the production of a polypeptide or its precursor. The polypeptide can be encoded by the full-length coding sequence or by any portion of the coding sequence such that the desired enzymatic activity is retained.

[0038] As described herein, the term "native" refers to the typical or wild-type form of a gene, gene product, or the properties of that gene or gene product when isolated from a naturally occurring source. In contrast, the terms "modified", "variant", "mutant", or "variant" refer to a gene or gene product that shows modification of sequence and functional properties (i.e., altered properties) compared to the native gene or gene product, and the modified gene or gene product has been genetically engineered and does not exist or occur naturally.

[0039] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence is inserted, which is introduced into a cell and which enables the cell to replicate it therein. The nucleic acid sequence can be "exogenous", which means that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell but is present at a location in the host cell nucleic acid where it is not normally present. Examples of vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, plant viruses) and artificial chromosomes (e.g., YACs). Those skilled in the art will have sufficient knowledge to construct vectors by standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0040] The term "expression vector" refers to any type of genetic construct that contains a nucleic acid coding that can be transcribed into RNA. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain various "control sequences", which refer to nucleic acid sequences necessary for the transcription and possibly translation of a coding sequence operably linked in a particular host cell. In addition to the control sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that serve other functions (e.g., antibiotic resistance, multiple cloning sites, etc.).

[0041] The term "therapeutically effective amount", as used herein, refers to the amount of a therapeutic composition or formulation (e.g., a modified CGL enzyme or a nucleic acid encoding such an enzyme) used in a method for achieving a therapeutic effect, i.e., depleting homocysteine in a patient's circulation to levels below normal reference values.

[0042] As used herein, the term "cystathionine-gamma-lyase" (CGL or cystathionase) refers to any enzyme that catalyzes the hydrolysis of cystathionine to cysteine. As used herein, the term also contemplates forms of primate cystathionine-gamma-lyase (or cystathionine-gamma-lyase), including forms of human cystathionine-gamma-lyase.

[0043] As used herein, the terms "treatment" and "treating" refer to the administration of a therapeutic agent to, or the application of a procedure or modality to, a subject for the purpose of obtaining a therapeutic benefit in a disease or health-related condition. For example, treatment may include the administration of a therapeutically effective amount of homocysteine (homocystinase).

[0044] As used herein, the terms "subject" and "patient" refer to any of a human or non-human, e.g., a primate, mammal, and vertebrate. The subject can be human.

[0045] Non-clinical dose range finding (DRF) studies Non-clinical experiments were conducted using pegylated cystathionine-gamma-lyase (CGL-ILMDRGVS-PEG) having the E59I, S63L, L91M, R119D, K268R, T311G, E339V, I353S mutations to characterize the pharmacology, PK, PD, safety, and toxicity of the compound. These studies support and enable the use of pegylated CGL-ILMDRGVS (CGL-ILMDRGVS-PEG) in a Phase 1 / 2 first-in-human clinical trial in subjects with homocystinuria due to CBS deficiency.

[0046] Deficiency of cystathionine beta-synthase (CBS) causes an increase in plasma levels of homocysteine and homocysteine analogs (such as homocystine and homocysteinylated peptides), and the combination of these represents total homocysteine (tHcy) in plasma. In in vivo animal studies, the intraperitoneal (IP) and subcutaneous (SC) administration routes are utilized to evaluate the pharmaceutical effect of polyhistidine-labeled CGL-ILMDRGVS-PEG (HIS-CGL-ILMDRGVS-PEG) on plasma tHcy levels in a clinically relevant knockout mouse model of classical homocystinuria.

[0047] In vivo animal studies were conducted in cynomolgus monkeys to characterize the pharmacokinetics (PK), pharmacodynamics (PD, tHcy depletion), and bioavailability utilizing both intravenous (IV) and subcutaneous (SC) administration routes of CGL-ILMDRGVS-PEG. Using these data, the non-clinical safety and toxicity (toxicity, toxicokinetics, and immunogenicity) of CGL-ILMDRGVS-PEG in normal Sprague-Dawley rats and cynomolgus monkeys were designed. Normal rats and monkeys are considered suitable for evaluating the sensitivity to CGL-ILMDRGVS-PEG toxicity because of their low plasma tHcy levels. This low plasma tHcy represents a more significant depletion of their blood tHcy levels than expected in homocystinuria patients.

[0048] The non-clinical safety profile of CGL-ILMDRGVS-PEG used to support the clinical development plan was also supported by dose range finding (DRF) studies after multiple IV or SC administrations of CGL-ILMDRGVS-PEG in rats and cynomolgus monkeys, and a planned 4-week Good Laboratory Practice (GLP) toxicity study in the same species. Rats were administered intravenously or subcutaneously once a week for 4 weeks starting on postnatal day 22, and cynomolgus monkeys (approximately 36 months old) were administered intravenously or subcutaneously once a week for 4 weeks. The 4-week toxicity study will establish the no-observed-adverse-effect level (NOAEL) necessary to support the initiation and duration of clinical administration in subjects 12 years of age and older.

[0049] To enable the clinical use of CGL-ILMDRGVS-PEG in pediatric patients under 12 years old (2 years or older to 11 years or younger), toxicity was evaluated in juvenile rats. In non-clinical trials using CGL-ILMDRGVS-PEG in the CBS- / - mouse disease model, it was shown that administration of HIS-CGL-ILMDRGVS-PEG could rescue neonatal lethality. Data obtained from non-clinical trials of HIS-CGL-ILMDRGVS-PEG and CGL-ILMDRGVS-PEG provide information to support the first human Phase 1 / 2 clinical trial in patients with homocystinuria due to CBS deficiency.

[0050] Table 2 summarizes the overall plan for supporting the IND, ongoing non-clinical trials, and non-clinical development plan.

Table 2

[0051] Non-clinical Pharmacology and Pharmacokinetics for DRF To show that homocysteine and engineered human CGL enzymes that break down homocysteine can reduce the toxic levels of tHcy in plasma, in vivo PD and efficacy studies were conducted using polyhistidine-tagged and / or PEGylated versions of the enzyme molecule CGL-ILMDRGVS (e.g., HIS-CGL-ILMDRGVS-PEG or CGL-ILMDRGVS-PEG). Additionally, CGL-ILMDRGVS-PEG was used in confirmatory PK / PD studies in cynomolgus monkeys and PD, and HIS-CGL-ILMDRGVS-PEG was used in efficacy studies in the CBS-deficient (CBS- / -) mouse model.

[0052] CBS- / - Mouse Model The CBS-deficient (CBS- / -) mouse model is a clinically relevant mouse model of classical homocystinuria (homocystinuria due to CBS deficiency). Using the CBS- / - mouse model, elevated plasma tHcy levels and downstream disease manifestations were evaluated. The CBS- / - mouse model is a clinically relevant mouse model of classical homocystinuria (homocystinuria due to CBS deficiency) that has many of the characteristics of human disease, including hyperhomocysteinemia, osteoporosis, and developmental disorders. Homozygous CBS- / - mice are neonatally lethal due to their genetic defect, but their lifespan can be extended by providing betaine, which remethylates homocysteine to methionine at an early age. However, betaine only improves survival in 50% of CBS- / - mice when given via breast milk up to the age of weaning, suggesting the need for more effective therapies.

[0053] In the study, CBS- / - mice were administered HIS-CGL-ILMDRGVS-PEG subcutaneously (SC) twice a week (BIW) at dose levels ranging from 3.125 to 25 mg / kg to determine whether a reduction in plasma tHcy levels in CBS- / - mice improves survival. Additionally, intraperitoneal (IP) administration at 25 mg / kg BIW and SC administration at 12.5 mg / kg once a week (QW) were tested. In this study, to enhance survival rates, all animals were able to ingest betaine via breast milk from birth until weaning. Administration of HIS-CGL-ILMDRGVS-PEG was initiated at 10 days of age and continued until at least 12 weeks of age when the animals were fully mature and the risk of survival impairment was no longer observed. A statistically significant improvement in survival was observed at all dose levels up to 3.125 mg / kg SC where no animal deaths were reported, compared to the PBS control (approximately 15% survival). Additionally, a clear improvement in liver pathology was observed (Figures 5, 6), indicating that HIS-CGL-ILMDRGVS-PEG is a superior therapy to betaine in CBS-deficient animals. Using the observed survival effect of HIS-CGL-ILMDRGVS-PEG in CBS- / - mice, a preliminary PD analysis was performed.

[0054] Pharmacokinetics and Pharmacodynamics Studies Using Monkeys Male cynomolgus monkeys were administered intravenous or subcutaneous doses of CGL-ILMDRGVS-PEG. The PK and PD characteristics of CGL-ILMDRGVS-PEG after IV or SC administration at 2 and 8 mg / kg were characterized by the study. Evaluation of the injection site after weekly SC administration for 3 weeks was conducted in the second phase of the study.

[0055] Total (free and bound) homocysteine and its oxidized form of homocysteine (referred to as total homocysteine (tHCY)) were used as pharmacodynamic markers. The mean clearance (CL) was slightly lower at low doses, but the range (mean ± SD) overlapped after IV administration. The observed volume of distribution (Vss) was almost equivalent to the monkey serum volume (45 mL / kg), but the mean estimated value tended to be slightly lower. The obtained mean IV half-life (T1 / 2) was in the range of 88.4 - 93.6 hours (about 3.7 - 3.9 days). Intravenous exposure (C max , AUC 0-∞ ) increased almost proportionally to the dose, and for a four-fold change in dose, an approximately 3.6-fold and 3.4-fold increase in mean C max and AUC 0-∞ were observed. The median of T max was observed at 0.5 hour after administration.

[0056] Subcutaneous exposure (C max , AUC 0-∞ ) was higher at 8 mg / kg compared to 2 mg / kg. The median of T max was 48 hours at 8 mg / kg compared to 24 hours after administration at 2 mg / kg. The mean bioavailability estimate at 8 mg / kg was moderate (75.8%). Bioavailability was not evaluated at the 2 mg / kg dose.

[0057] After intravenous administration of CGL-ILMDRGVS-PEG, the maximum decrease in plasma tHCY levels was observed 4 to 8 hours after administration, and further average decreases were observed at a high intravenous administration level of 8 mg / kg. Five minutes after administration (2 and 8 mg / kg, respectively), there were initial decreases of approximately 39% and approximately 72% in plasma tHCY levels, after which they gradually decreased to the maximum suppression observed. The average tHCY levels 8 hours after administration were 47.8% and 18.1% of the average baseline levels (corresponding to decreases of 52.2% and 81.9%, respectively). This corresponded to average CGL-ILMDRGVS-PEG concentrations of 58.6 and 194 μg / mL or more, respectively. Recovery to baseline tHCY levels was almost complete by 240 hours in the 2 mg / kg intravenous administration group and ~84% was complete by 336 hours (2 weeks) in the 8 mg / kg intravenous administration group.

[0058] After subcutaneous administration of CGL-ILMDRGVS-PEG, at the 8 mg / kg dose level, the maximum decrease in plasma tHCY levels was observed 24 to 48 hours after administration, and at the 2 mg / kg dose, 8 to 24 hours after administration. The tHCY levels decreased steadily until the maximum decrease was obtained. The average tHCY levels at the maximum decrease were 75.5% and 42.0% of the average baseline levels (corresponding to decreases of 24.5% and 58.0%, respectively) at the 2 and 8 mg / kg doses. This corresponded to average CGL-ILMDRGVS-PEG concentrations of 34.9 and 98.7 μg / mL, respectively, and it should be noted that there was significant variation among animals at 2 mg / kg. Recovery to baseline tHCY levels was complete by 336 hours or on day 15 (2 weeks) of administration for the 8 mg / kg / day 1 dose. After administration of 2 mg / kg on day 15, recovery appeared to be complete by 168 hours after administration.

[0059] Dosage and Administration Accordingly, one or more embodiments of the present invention provide a dosing regimen for a modified CGL enzyme, such as CGL-ILMDRGVS or CGL-ILMDRGVS-PEG. In various embodiments, the enzyme is administered at a dose of about 0.05 mg / kg to about 4 mg / kg, such as about 0.05 mg / kg, about 0.1 mg / kg, about 0.15 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, about 0.5 mg / kg, about 0.5 mg / kg, about 0.55 mg / kg, about 0.6 mg / kg, about 0.65 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.85 mg / kg, about 0.9 mg / kg, about 0.95 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, or about 4 mg / kg. For a PEGylated enzyme (e.g., CGL-ILMDRGVS-PEG), the enzyme dose is calculated based on the non-PEGylated form of the enzyme (e.g., CGL-ILMDRGVS).

[0060] In one or more embodiments, the modified CGL enzyme (e.g., CGL-ILMDRGVS or CGL-ILMDRGVS-PEG) (or a composition comprising such an enzyme) is administered in multiple doses. In various embodiments, the enzyme is administered once a day to once a month, such as once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, or once a month.

[0061] Exemplary dosing regimens include from about 0.05 mg / kg to about 4 mg / kg administered at a frequency of once a day to once a month. In one or more embodiments, the enzyme is administered at a dose of 0.1 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.15 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.2 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.25 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.3 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.35 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.4 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.45 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.5 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.6 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.7 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.8 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 0.9 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 1 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 1.5 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 2 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 2.5 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 3 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 3.5 mg / kg once a week. In one or more embodiments, the enzyme is administered at a dose of 4 mg / kg once a week.

[0062] The modified CGL enzyme (or composition comprising such an enzyme) described herein can be administered via any suitable route including, but not limited to, intravenous, intrathecal, subcutaneous, intramuscular, intratumoral, and / or intraperitoneal. In one or more embodiments, the modified CGL enzyme (or composition comprising such an enzyme) is administered intravenously (IV) or subcutaneously (SC).

[0063] Cystathionine-gamma-lyase Lyases are enzymes that catalyze the cleavage of various chemical bonds, often forming a new double bond or a new ring structure. For example, the enzyme catalyzing this reaction could be a lyase such as: ATP-----+cAMP+PPi. Lyases differ from other enzymes in that they require only one substrate for the forward reaction, but two substrates for the reverse reaction.

[0064] Many pyridoxal-5'-phosphate (PLP)-dependent enzymes are involved in the metabolism of cysteine, homocysteine, and methionine, and these enzymes form an evolutionarily related family called Cys / Met metabolism PLP-dependent enzymes. These enzymes are proteins of approximately 400 amino acids, and the PLP group is bound to a lysine residue located at the central position of the polypeptide. Members of this family include cystathionine-gamma-lyase (CGL), cystathionine-gamma-synthase (CGS), cystathionine-beta-lyase (CBL), methionine-gamma-lyase (MGL), and O-acetylhomoserine (OAH) / O-acetylserine (OAS) sulfhydrylase (OSHS). Common to all of these is the formation of a Michaelis complex that results in an external substrate aldimine. The further course of the reaction is determined by the substrate specificity of the particular enzyme.

[0065] For example, specific mutations were introduced into a PLP-dependent lyase family member, such as human cystathionine-γ-lyase, to alter its substrate specificity. In this way, a novel variant with de nova ability to degrade homocysteine (homocysteine) as a substrate and having a catalytic activity higher than that of hGGL-NLV was created. Modification of other PLP-dependent enzymes to generate a novel homocysteine (homocysteine) degrading activity is also contemplated.

[0066] CGL is a tetramer that catalyzes the final step in the mammalian sulfur transfer pathway (Ra et al., 1990). CGL catalyzes the conversion of L-cystathionine to L-cysteine, alpha-ketobutyrate, and ammonia. Pyridoxal phosphate is a cofactor family of this enzyme. Using protein engineering, cystathionase, which has only weak activity against the degradation of homocysteine and homocystine, was converted into an enzyme that can rapidly degrade homocysteine and homocystine (U.S. Patent No. 9,481,877, which is hereby incorporated by reference in its entirety).

[0067] Homocysteine (homocysteine) engineering Since humans do not produce homocystinase (homocysteinase), it is necessary to engineer homocystinase (homocysteinase) for human therapy that has high activity and specificity for degrading homocysteine (homocysteine) under physiological conditions and high stability in physiological fluids such as serum, and they are also non-immunogenic because they are natural proteins that usually induce immune tolerance.

[0068] Due to the undesirable immunogenic effects seen in animal studies using pMGL (MGL from P. putida), it is desirable to engineer homocysteine (homocysteine) degrading activity in human enzymes. Immune tolerance to human proteins allows such enzymes to be non-immunogenic or have minimal immunogenicity and thus become sufficiently tolerated.

[0069] Mammals do not have homocystinase (homocysteine lyase), but have cystathionine-γ-lyase (CGL). CGL is a tetramer that catalyzes the final step in the mammalian sulfur transfer pathway (Rao et al., 1990). CGL catalyzes the conversion of L-cystathionine to L-cysteine, alpha-ketobutyrate, and ammonia. The cDNA of human CGL (hCGL) has been cloned and expressed in the past, but the yield was relatively low (about 5 mg / L culture) (Lu et al., 1992; Steegbom et al., 1999).

[0070] Accordingly, methods and compositions are provided related to modifying primate (particularly human) cystathionine-γ-lyase (CGL or cystathionase) via mutagenesis to efficiently hydrolyze homocysteine (homocysteine).

[0071] Modified CGL enzymes are described that exhibit at least one functional activity comparable to that of the unmodified CGL enzyme. The modified CGL enzyme can be further modified to increase serum stability. Examples of modified CGL enzymes include proteins that have additional advantages such as homocystinase (homocysteine lyase) enzyme activity compared to the unmodified CGL enzyme. The unmodified protein or polypeptide can be a native cystathionine-γ-lyase such as human cystathionine-γ-lyase.

[0072] Measurement of activity can be carried out using assays well known to those skilled in the art, particularly with respect to enzyme activity, and for comparison purposes can include the use of natural and / or recombinant forms of either the modified or unmodified enzyme. For example, homocystinase (homocysteine lyase) activity can be determined by any assay for detecting the production of any substrate resulting from the conversion of homocysteine (homocysteine) such as alpha-ketobutyrate, methanethiol, and / or ammonia.

[0073] Modified CGL enzymes can be identified based on an increase in homocysteine degradation activity. For example, the substrate recognition site of an unmodified polypeptide can be identified. This identification can be performed based on structural analysis or homology analysis. A population of variants containing modifications to such a substrate recognition site can be generated. Variants with increased homocysteine degradation activity can be selected from the variant population. The selection of the desired variant can include methods such as the detection of by-products or products from homocysteine degradation.

[0074] The modified CGL enzyme can have amino acid deletions and / or substitutions. In other words, an enzyme with a deletion, an enzyme with a substitution, and an enzyme with both a deletion and a substitution are modified CGL enzymes. These modified CGL enzymes can further contain inserted or added amino acids and can be, for example, fusion proteins or proteins with linkers. A "modified deletion CGL enzyme" lacks one or more residues of the native enzyme but can have the specificity and / or activity of the native enzyme. The modified deletion CGL enzyme can also have reduced immunogenicity or antigenicity. An example of a modified deletion CGL enzyme is one in which amino acid residues are deleted in at least one antigenic region, i.e., the enzyme region determined to be antigenic in a specific organism such as the type of organism to which the modified CGL enzyme can be administered.

[0075] A substitution or substitution variant may involve the replacement of one amino acid with another at one or more sites within a protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector function and / or bioavailability. The substitution may be conservative or non-conservative, i.e., one amino acid is replaced with one having a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the substitution of alanine with serine, arginine with lysine, aspartic acid or histidine, glutamic acid, cysteine with glutamine, glutamine with asparagine, glutamic acid with asparagine, glycine with proline, histidine with aspartic acid or glutamine, isoleucine with leucine, leucine or isoleucine, lysine with arginine, methionine or isoleucine, phenylalanine with tyrosine, serine with threonine, threonine to serine, tryptophan with tyrosine, tyrosine with tryptophan or phenylalanine, and valine with isoleucine or leucine.

[0076] In addition to deletions or substitutions, the modified CGL enzyme may typically have an insertion of residues, which may include the addition of at least one residue in the enzyme. This may include the insertion of a targeting peptide or polypeptide, or simply a single residue. Terminal additions, called fusion proteins, are described below.

[0077] The term "biological functional equivalent" is well understood in the art and is further defined in more detail herein. Accordingly, a CGL enzyme sequence having at least about 90% sequence identity to SEQ ID NO:1, or an amino acid sequence having about 91% to about 99% (including 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) amino acid identity or such conservative substitutions to the amino acids of the 10 modified CGL enzymes disclosed herein is included, provided that the biological activity of the enzyme is such that 15 measurable biological activity parameters (e.g., conversion of homocysteine to alpha-ketobutyrate, methanethiol, and ammonia) are about 20%, about 15%, about 10%, or about 5% of the modified CGL enzymes disclosed herein. The modified CGL enzymes can be biologically functionally equivalent to their unmodified counterparts.

[0078] Amino acid and nucleic acid sequences can include additional N-terminal or C-terminal amino acids or additional residues such as 5' or 3' sequences, and still be essentially as described for one of the sequences disclosed herein, provided that the sequence meets the above criteria such as maintaining the biological protein activity involved in protein expression. The addition of terminal sequences is effected, in particular, by applying to the nucleic acid sequence various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or various internal sequences known to be present within the gene, i.e., introns.

[0079] Conjugate The provided compositions and methods include further modification of the modified CGL enzyme for improvement, such as by forming conjugates with polymers such as heterologous peptide segments or polyethylene glycol. The modified CGL enzyme can be linked to PEG to increase the hydrodynamic radius of the enzyme and thus increase its persistence in serum. The disclosed polypeptides can be conjugated to any targeting agent, such as a ligand that has the ability to specifically and stably bind to an external receptor or binding site on tumor cells (U.S. Patent Publication No. 2009 / 0304666). PEG can be of a size from about 3,000 to 20,000 daltons, and an exemplary size is about 5,000 daltons.

[0080] Fusion protein Fusion proteins are provided in which the modified CGL enzyme can be linked to a heterologous domain at the N-terminus or C-terminus. For example, in the fusion, a leader sequence from another species can also be employed to enable recombinant expression of the protein in a heterologous host. Another useful fusion is the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or preferably an immunologically active domain such as a cleavable antibody epitope, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0081] The modified CGL enzyme can be linked to a peptide that increases the in vivo half-life, such as an XTEN polypeptide (Schellenberger et al., 2009), an IgG Fe domain, albumin, or an albumin-binding peptide.

[0082] Methods for generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein or by ligation of DNA sequences encoding the heterologous domains followed by expression of the intact fusion protein.

[0083] The production of a fusion protein that restores the functional activity of a parent protein can be facilitated by ligating a gene with a crosslinking DNA segment encoding a peptide linker that is spliced between tandemly linked polypeptides. The linker is of sufficient length to allow proper folding of the resulting fusion protein.

[0084] Linker The modified CGL enzyme may be chemically conjugated using a bifunctional crosslinking reagent or fused at the protein level with a peptide linker. Bifunctional crosslinking reagents are widely used for a variety of purposes, including the preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structure-related studies. A suitable peptide linker, such as a Gly-Ser linker, can also be used to ligate the modified CGL enzyme.

[0085] Homobifunctional reagents having two identical functional groups can induce crosslinking between identical and different macromolecules or subunits of a macromolecule and ligate polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By utilizing the reactivity of each of the two functional groups, crosslinking can be controlled in both selectivity and continuity. Bifunctional crosslinking reagents can be classified according to the specificity of their functional groups, such as amino-, sulfhydryl-, guanidine-, indole-, carboxyl-specific groups, etc. Among these, reagents targeting free amino groups have become common due to their commercial availability, ease of synthesis, and mild reaction conditions under which they can be applied.

[0086] Some hetero-bifunctional crosslinking reagents contain a primary amine-reactive group and a thiol-reactive group. In another example, hetero-bifunctional crosslinking reagents and methods of using crosslinking reagents are described in the literature (U.S. Patent No. 5,889,155, which is specifically incorporated herein by reference in its entirety). The crosslinking reagent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue and, in one example, enables the coupling of an aldehyde to a free thiol. The crosslinking reagent can be modified to crosslink various functional groups.

[0087] Additionally, any other linking / coupling agent and / or mechanism known to those of skill in the art, such as antibody-antigen interactions, avidin-biotin binding, amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, phosphoester bonds, phosphoramide bonds, anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof, can be used to combine the modified CGL.

[0088] It is preferred to use a crosslinking agent that has reasonable stability in the blood. A number of types of disulfide bond-containing linkers are known that can be successfully used to conjugate targeting agents and therapeutic / preventive agents. Linkers containing sterically hindered disulfide bonds have been shown to confer higher stability in vivo. Thus, these linkers are one type of linker.

[0089] In addition to hindered crosslinking agents, unhindered linkers can also be used according to the present disclosure. Other useful crosslinking agents that are thought not to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such crosslinking agents is well understood in the art. Flexible linkers can also be used.

[0090] When chemically conjugated, peptides are generally purified to separate the conjugate from unconjugated agents and other contaminants. A number of purification techniques are available and, by using them, a conjugate of sufficient purity to have clinical utility can be obtained.

[0091] Purification methods based on size separation, such as gel filtration, gel permeation, or high performance liquid chromatography, are generally the most useful. Other chromatography techniques, such as Blue-Sepharose separation, may also be used. Conventional methods for purifying fusion proteins from inclusion bodies, such as using a mild detergent like sodium N-lauroyl sarcosinate (SLS), may be useful.

[0092] PEGylation Methods and compositions related to the PEGylation of modified CGL enzymes are disclosed. For example, a modified CGL enzyme can be PEGylated according to the methods disclosed herein.

[0093] PEGylation is the process of covalently attaching a poly(ethylene glycol) polymer chain to another molecule, usually a drug or therapeutic protein. "PEGylation" is typically carried out by incubating a reactive derivative of PEG with the target macromolecule. Covalent attachment of PEG to a drug or therapeutic protein can increase the hydrodynamic size (size in solution) of the agent so as to "mask" the agent from the host's immune system (reducing immunogenicity and antigenicity) and extend its circulation time by decreasing renal clearance. PEGylation can also confer water solubility to hydrophobic drugs and proteins.

[0094] The first step in PEGylation is the suitable functionalization of the PEG polymer at one or both ends. PEG activated at each end with the same reactive moiety is known as "homo-bifunctional", while if the functional groups present are different, the PEG derivative is termed "hetero-bifunctional" or "heterofunctional". Chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

[0095] The selection of suitable functional groups for the PEG derivative is based on the type of available reactive groups on the molecule to which the PEG will be attached. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used.

[0096] The techniques used to form first-generation PEG derivatives generally involve reacting the PEG polymer with groups that react with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In second-generation PEGylation chemistry, more efficient functional groups such as aldehydes, esters, and amides are available for conjugation.

[0097] As the uses of PEGylation become increasingly developed and complex, the need for hetero-bifunctional PEG for conjugation is increasing. Hetero-bifunctional PEG is very useful for linking two entities when a hydrophilic, flexible, and biocompatible spacer is required. Preferred end groups for hetero-bifunctional PEG are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0098] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. These activities rely on adding protein modification groups to the alcohol terminus. In some examples, polyethylene glycol (PEG diol) is used as the precursor molecule. The diol is then modified at both ends to generate hetero- or homo-dimeric PEG linker molecules.

[0099] Proteins are generally PEGylated at nucleophilic sites such as unprotonated thiols (cysteiny residues) or amino groups. Examples of cysteine-specific modification reagents include PEG maleimide, PEG iodoacetic acid, PEG thiol, and PEG vinyl sulfone. All four exhibit strong cysteine specificity under mild conditions and at neutral to weakly alkaline pH, but each has some drawbacks. The thioether formed with maleimide can be somewhat unstable under alkaline conditions, so there may be some limitations to formulation options using this linker. The carbamothioate bond formed with iodo-PEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms a disulfide bond with the protein thiol, but this bond can also be unstable under alkaline conditions. The PEG-vinyl sulfone reactivity is relatively slow compared to maleimide and iodo-PEG, however, the thioether bond formed is extremely stable. The slow reaction rate also makes the control of the PEG-vinyl sulfone reaction easier.

[0100] Site-specific PEGylation at native cysteine residues is rarely done because these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis methods can be used to incorporate cysteine PEGylation sites for thiol-specific linkers. The cysteine mutations must be designed such that they are available to the PEGylation reagent and remain biologically active after PEGylation.

[0101] Amino-specific modifiers include PEG NHS esters, PEG tresylates, PEG aldehydes, PEG isothiocyanates, and several others. All react under mild conditions and are very specific for amino groups. PEG NHS esters are perhaps one of the more reactive agents, however, their high reactivity can make control of large-scale PEGylation reactions difficult. PEG aldehydes form imines with amino groups and are then reduced to secondary amines with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and becomes volatile, especially at lower pH, and must be handled with care.

[0102] Since most proteins have multiple lysine residues, site-specific PEGylation can be difficult. Fortunately, these reagents react with unprotonated amino groups, so by performing the reaction at a lower pH, it is possible to PEGylate the lower pK amino groups. Generally, the pK of the alpha-amino group is 1-2 pH units lower than that of the epsilon-amino group of lysine residues. By PEGylating the molecule below pH 7, high selectivity for the N-terminus can often be achieved. However, this is only possible if the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits of PEGylation often outweigh a significant loss of in vitro biological activity, resulting in products with much greater in vivo biological activity regardless of the chemistry of the PEGylation.

[0103] When developing a PEGylation procedure, there are several parameters to consider. Fortunately, there are usually four or fewer parameters. A "design of experiments" approach to optimizing PEGylation conditions can be very useful. For a thiol-specific PEGylation reaction, the parameters to consider include protein concentration, PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time, and in some cases, exclusion of oxygen. (Oxygen can cause intermolecular disulfide formation by the protein and will reduce the yield of the PEGylated product.) For amine-specific modification, the same factors (except for oxygen) should be considered, except that pH can be even more decisive, especially when targeting the N-terminal amino group.

[0104] For both amine-specific and thiol-specific modifications, the reaction conditions can affect the stability of the protein. This can limit the temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before initiating the PEGylation reaction. For example, if the activity of the PEGylation agent is only 70%, the amount of PEG used should ensure that only the active PEG molecules are counted in the stoichiometry of the protein-PEG reaction.

[0105] Proteins and Peptides Compositions are provided that include at least one protein or peptide (e.g., a modified CGL enzyme). These peptides can be included in a fusion protein or conjugated to an agent, as described above.

[0106] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full-length sequence translated from a gene, a polypeptide of about 100 or more amino acids, and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0107] Accordingly, the term "protein or peptide" encompasses an amino acid sequence containing at least one of the 20 common amino acids found in naturally occurring proteins or at least one non-natural amino acid.

[0108] Proteins or peptides can be produced by any technique known to those skilled in the art, such as the expression of proteins, polypeptides or peptides by standard molecular biology techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides and peptides are known to those skilled in the art.

[0109] Nucleic Acids and Vectors Nucleic acid sequences encoding modified CGL enzymes or fusion proteins containing modified CGL enzymes are disclosed. Depending on the expression system used, the nucleic acid sequence can be selected based on conventional methods. For example, if a modified CGL enzyme is derived from human cystathionase and contains multiple codons that are not first utilized in E. coli, it can be hampered in expression. Thus, each gene or its variant can be codon-optimized for E. coli expression using free software (see Hoover and Lubkowski (2002)) to design the coding sequence to have no rare codons. Various vectors can be used to express proteins of interest such as modified CGL. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0110] Host Cells The host cell can be transformed to enable the expression and secretion of the modified CGL enzyme and its conjugate. The host cell can be a bacterium, a mammalian cell, a yeast, or a filamentous fungus. Various bacteria include Escherichia and Bacillus. Yeasts belonging to Saccharomyces, Kiuyveromyces, Hansenula, or Pichia can be used as suitable host cells. Various types of filamentous fungi containing the following genera can be used as expression hosts: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, and Pyricularia.

[0111] Examples of host organisms that can be used include bacteria such as derivatives of Escherichia coli MC1061, Bacillus subtilis BRB1 (Sibakov et al., 1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcus lividans (Hopwood et al., 1985), yeasts such as Saccharomyces cerevisiae AH 22 (Mellor et al., 1983) or Schizosaccharomyces pombe, and filamentous fungi such as Aspergillus nidulans, Aspergillus awamori (Ward, 1989), or Trichoderma reesei (Penttila et al., 1987; Harkki et al, 1989).

[0112] Examples of mammalian host cells include Chinese hamster ovary (CHO-K1, ATCC number CCL61), rat pituitary cells (GH1, ATCC number CCL82), human cervical cells (HeLa S3, ATCC number CCL2.2), rat hepatocarcinoma cells (H-4-II-E, ATCC number CRL-1548), SV40-transformed MK cells (COS-1, ATCC number CRL-1650), and mouse embryonic fibroblasts (NIH-3T3, ATCC number CRL-1658). The foregoing is meant to exemplify, but not limit, many possible host organisms known in the art. In principle, any host that can secrete, whether prokaryotic or eukaryotic, can be used.

[0113] Mammalian host cells expressing CGL enzymes and / or their fusion proteins are cultured under conditions typically used for culturing the parental cell line. Generally, the cells are cultured in a standard culture medium containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM, or DMEM, typically supplemented with 5-10% serum, such as fetal bovine serum. The culture conditions are also standard, for example, incubating the culture at 37°C in a stationary or roller culture vessel until the desired level of protein is obtained.

[0114] Protein Purification Protein purification techniques are well known to those skilled in the art. These techniques, at one level, require homogenization and crude fractionation of the polypeptide and non-polypeptide fractions of cells, tissues or organs. The protein or polypeptide of interest can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity), unless otherwise specified. Analytical methods particularly suitable for the preparation of pure peptides are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. Particularly efficient methods for purifying peptides are fast performance liquid chromatography (FPLC), or even more so, high performance liquid chromatography (HPLC).

[0115] A purified protein or peptide is intended to refer to a composition that can be isolated from other components, where the protein or peptide is purified to any degree relative to its naturally available state. Thus, an isolated or purified protein or peptide also refers to a protein or peptide that has been freed from the environment in which it may naturally occur. Generally, "purified" refers to a protein or peptide composition that has been subjected to fractionation to remove various other components and substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, for example, a composition in which the protein constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the composition.

[0116] A variety of techniques suitable for use in protein purification are well known to those skilled in the art. These include, for example, precipitation using ammonium sulfate, PEG, and antibodies, or heat denaturation, followed by centrifugation, ion exchange, gel filtration, reverse phase, hydroxylapatite, and affinity chromatography steps such as isoelectric focusing, gel electrophoresis, and combinations of these techniques with other techniques. As is generally known in the art, the order in which various purification steps are performed may be changed or a particular step may be omitted, and still result in a method suitable for the preparation of a substantially purified protein or peptide, it is believed.

[0117] A variety of methods for quantifying the degree of purification of a protein or peptide are known to those skilled in the art. These include, for example, determining the specific activity of the active fraction or assessing the amount of polypeptide in the fraction by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS / PAGE) analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it to the specific activity of the initial extract, and thus calculate the purity therein as evaluated by the "fold purification value". The actual units used to represent the amount of activity, of course, depend on the particular assay technique selected after purification and whether the expressed protein or peptide exhibits a detectable activity.

[0118] The requirement that a protein or peptide should always be provided in its most purified state is not usual. In fact, products that are substantially less purified can be considered useful. Partial purification can be achieved by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, cation exchange column chromatography performed using a high performance liquid chromatography (HPLC) apparatus is generally understood to provide a greater "multiple" of purification than the same technique using a low pressure chromatography system. Methods showing a lower relative degree of purification can have advantages in the total recovery of the protein product or in the maintenance of the activity of the expressed protein.

[0119] The protein or peptide may be isolated or purified, for example, as a modified CGL enzyme, a fusion protein containing a modified CGL enzyme, or a modified CGL enzyme after PEGylation. For example, to facilitate purification, a His-tag or an affinity epitope can be included in such a modified CGL enzyme. Affinity chromatography is a chromatographic procedure that relies on the specific affinity between the substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type interaction. The column material is synthesized by covalently bonding one of the binding partners to an insoluble matrix. The column material can then specifically adsorb the substance from the solution. Elution occurs by changing the conditions so that binding does not occur (for example, changes in pH, ionic strength, temperature, etc.). The matrix must be a substance that does not adsorb molecules to a significant extent and has a wide range of chemical, physical, and thermal stability. The ligand should be bonded in such a way that it does not affect its binding properties. The ligand should also provide a relatively strong bond. It must be possible to elute the substance without destroying the sample or the ligand.

[0120] Size exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is typically applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, this technique is known as gel filtration chromatography as opposed to gel permeation chromatography, which uses an organic solvent as the mobile phase.

[0121] The basic principle of SEC is that particles of different sizes elute (are filtered) through the stationary phase at different rates. This separates the solution of particles based on size. If all the particles are loaded simultaneously or almost simultaneously, particles of the same size should elute together. Each size exclusion column has a range of molecular weights that it can separate. The exclusion limit is the point where the molecular weight at the upper end of this range is defined, where molecules are too large to be captured by the stationary phase. The permeation limit defines the molecular weight at the lower end of the separation range, where molecules of a sufficiently small size can fully penetrate the pores of the stationary phase, and all molecules below this molecular mass elute as a single band because they are very small.

[0122] High performance liquid chromatography (or high pressure liquid chromatography, HPLC) is a form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds a packing material for chromatography (stationary phase), a pump to move the mobile phase through the column, and a detector that indicates the retention time of the molecules. The retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent used.

[0123] Before describing some exemplary embodiments of the present disclosure, it should be understood that the present disclosure is not limited to the details of the configurations or process steps described below. The present disclosure is capable of other embodiments and can be practiced or implemented in various ways.

Examples

[0124] The following examples are included to illustrate preferred specific examples of the present invention. The techniques disclosed in the following examples represent techniques that have been found by the inventors to function well in the practice of the present invention, and thus, it should be understood by those skilled in the art that they also function well in the practice of the present invention. However, those skilled in the art will understand that, in view of the disclosure of the present invention, many modifications can be made in specific examples that are disclosed and that yield similar results without departing from the spirit and scope of the present invention.

[0125] Example 1: Rescue of CBS− / − Mice by Engineered Human Cystathionine-γ-Lyase CBS-deficient (CBS− / −) mice are used as an animal model of human homocystinuria due to CBS deficiency. The physiological presentation is similar to human disease in that it shows elevated levels of plasma tHcy in the mice. Intraperitoneal (IP) and subcutaneous (SC) administrations of HIS-CGL-ILMDRGVS-PEG were evaluated for various properties such as the ability to enhance the viability of CBS− / − mice. The mice were fed a normal (methionine-containing) diet and thus also provided a model for ad libitum feeding among human patients.

[0126] When the physical health deteriorated (e.g., due to illness), the mice were euthanized and this was recorded as the survival time. CBS− / − mice were administered HIS-CGL-ILMDRGVS-PEG at doses in the range of 3.125 - 25 mg / kg by SC twice a week (BIW) to determine whether a decrease in plasma tHcy levels in CBS− / − mice improved survival (Table 3 and FIG. 4). [Table 3]

[0127] Assuming that the human equivalent dose is about 1 / 12 of the mouse dose, the doses in this study are in the range of about 0.13 mg / kg to about 2 mg / kg of human equivalent dose.

[0128] Depending on the experiment, neonatal CBS− / − mice were allowed to ingest betaine via breast milk from birth to weaning to enhance survival, thereby ensuring sufficient mobilization of animals for the experiment. Administration with HIS-CGL-ILMDRGVS-PEG or CGL-ILMDRGVS-PEG was initiated at 10 days of age and continued until the animals reached at least 10–12 weeks of age when they were fully mature and the risk of survival impairment was no longer observed. A statistically significant improvement in survival was observed at all dose levels up to 3.125 mg / kg SC where no animal deaths were reported, compared to PBS control (∼15% survival) (Figure 4). In addition, a marked improvement in liver pathology was observed (Figures 6–8), suggesting that the modified CGL enzymes (HIS-CGL-ILMDRGVS-PEG and CGL-ILMDRGVS-PEG) are superior therapies to betaine in animals with CBS deficiency.

[0129] When administered TIW with 1.56 mg / kg of HIS-CGL-ILMDRGVS-PEG or BIW with PBS over 16 weeks, survival rates of 40% and 12% were obtained, respectively, whereas when HIS-CGL-ILMDRGVS-PEG was administered BIW or QW, survival rates greater than 50% were obtained at all dose levels tested (Figure 4). The survival rate of mice administered BIW with 12.5 or 3.125 mg / kg was 100%, and that of mice administered BIW with 6.25 mg / kg was 83%. The median survival period was 3 weeks with PBS administration and 6.75 weeks with TIW 1.56 mg / kg HIS-CGL-ILMDRGVS-PEG.

[0130] As the disease progresses, pathology can be observed in the organs and tissues of CBS - / - mice. Liver samples were taken from healthy wild - type mice (Figure 6A) or untreated CBS - / - mice and stained with hematoxylin and eosin (H&E). In 10 - day - old untreated mice, microvascular steatosis and the presence of lipid droplets were seen (Figure 6B and Figure 7A). Liver samples taken from 23 - day - old (Figure 6C) and 21 - day - old (Figure 7B) PBS - treated mice and stained with H&E showed higher microvascular steatosis, necrosis, lipid droplets, and macro - vascular steatosis compared to samples from 10 - day - old untreated mice, as well as samples from 23 - day - old (Figure 6D) treated mice or 21 - day - old mice treated with either 6.25 or 12.5 mg / kg of HIS - CGL - ILMDRGVS - PEG BIW (Figure 7C, D). Liver samples taken from 60 - day - old mice treated with HIS - CGL - ILMDRGVS - PEG showed disease reversal (Figure 6E).

[0131] Liver samples were also stained with Oil - red - O. Oil - red - O staining is a lysochromic (lipid - soluble dye) diazo dye used for the staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. In histology, a supersaturated solution of Oil - red - O in isopropanol can be used to stain fat in tissues.

[0132] Liver samples taken from 10 - day - old untreated mice, 19 - 21 - day - old PBS - treated mice, and 20 - day - old mice TIW - treated with 1.56 mg / kg of HIS - CGL - ILMDRGVS - PEG were stained with Oil - red - O. All of these mice showed a significant increase in Oil - red - O staining compared to samples taken from 21 - day - old mice BIW - treated with 12.5 mg / kg of HIS - CGL - ILMDRGVS - PEG, suggesting a decrease in neutral triglycerides, lipids, and / or lipoproteins in the livers of mice administered 12.5 mg / kg of HIS - CGL - ILMDRGVS - PEG BIW (Figure 8A, B, C, and D).

[0133] The effects on hair loss and osteoporosis were measured in CBS− / − mice treated SC BIW with 10 mg / kg of CGL-ILMDRGVS-PEG. The degree of hair loss was significantly decreased in CGL-ILMDRGVS-PEG-treated CBS− / − mice compared to the vehicle control group on day 170 (Figure 9A). The level of bone mineral density (BMD) was significantly higher in CGL-ILMDRGVS-PEG-treated CBS− / − mice compared to the vehicle control group on day 169 (Figure 9B).

[0134] Total homocysteine (tHcy) was measured in plasma, liver, and brain samples of vehicle-administered wild-type and CBS− / − mice and CGL-ILMDRGVS-PEG-administered CBS− / − mice (Figure 10A, B, and C). All mice were treated subcutaneously 3 times BIW. Vehicle-administered CBS− / − mice showed a significant increase in plasma, liver, and brain tHcy levels compared to the wild-type group. Treatment in CBS− / − mice with 10 mg / kg of CGL-ILMDRGVS-PEG significantly decreased tHcy levels in plasma, liver, and brain samples compared to the vehicle-treated group, suggesting an effective effect of using CGL engineered as a potential therapy for homocystinuria.

[0135] Pharmacodynamic analysis was performed using the survival effect of HIS-CGL-ILMDRGVS-PEG observed in CBS− / − mice. In the CBS− / − tests described in the next section, the "maintenance" dosing of the animals was in the range of 5 - 10 weeks.

[0136] Example 2: Single-Dose Pharmacodynamic Test in CBS− / − Mice The pharmacodynamic (PD) effect of HIS-CGL-ILMDRGVS-PEG was evaluated by administration to CBS− / − mice. CBS− / − mice are an animal model of human homocystinuria due to CBS deficiency that exhibit high plasma tHcy levels. CBS− / − mice that had demonstrated survival over 12 weeks (and thus were no longer at risk of neonatal lethality) were removed from HIS-CGL-ILMDRGVS-PEG administration (multiple dose levels), given a 25-day washout period to remove residual drug, and returned to a baseline level of abnormally high plasma tHcy. During this time, the animals were closely monitored for deteriorating clinical signs resulting from increased circulating tHcy. The mice were then randomized into two groups and administered a single subcutaneous (SC) dose of 12.5 or 6.25 mg / kg of HIS-CGL-ILMDRGVS-PEG. Blood was collected at multiple time points over 2 weeks. The initial plasma tHcy concentration was approximately 250 μM, and at the first collection time point 24 hours after dosing, a maximal inhibition (decrease to approximately 100 μM) was observed for both groups. By 72 hours after dosing, the plasma tHcy levels in the 6.25 mg / kg dosing group had returned to baseline levels and were maintained for the remainder of the study. The plasma tHcy levels in the 12.5 mg / kg dosing group remained at approximately 100 μM until 72 hours and returned to baseline values by 168 hours (Figure 11).

[0137] (CBS− / mice) Further analysis of PD showed pre-dose plasma tHcy levels of an average of 257 μM, which decreased to an average of 107.3 μM and 132.4 μM 24 hours after a single dose of 12.5 or 6.25 mg / kg of HIS-CGL-ILMDRGVS-PEG, respectively, 72 hours after dosing. Plasma tHcy returned to baseline in animals dosed with 6.25 mg / kg of HIS-CGL-ILMDRGVS-PEG but was maintained at 153.7 μM in the group dosed with 12.5 mg / kg (Figure 11). At 168 hours after dosing, tHcy had returned to baseline in both groups.

[0138] Prior to administration, the plasma methionine levels in CBS− / − mice were (on average) 102 μM. A dose of 6.25 mg / kg did not reduce methionine levels over a 336-hour time course (Figure 12). After 72 hours of administration of 12.5 mg / kg of HIS-CGL-ILMDRGVS-PEG, the plasma methionine levels decreased to an average of 78 μM (a 24% decrease). By 168 hours, the methionine levels were 82 μM, and by 240 hours they had returned to baseline (Figure 12). Plasma total methionine levels after single SC administration of 12.5 mg / kg or 6.25 mg / kg of HIS-CGL-ILMDRGVS-PEG.

[0139] Example 3: Multiple-dose pharmacodynamics study in CBS− / − mice CBS− / − mice that had demonstrated survival over 12 weeks were removed from HIS-CGL-ILMDRGVS-PEG administration (multiple-dose levels) and given a 15-day washout period to remove residual drug and return their tHcy levels to abnormally high baseline levels. After the washout period, the mice were randomized into three groups and administered HIS-CGL-ILMDRGVS-PEG at 12.5 or 6.25 mg / kg SC twice a week (BIW) or at 12.5 mg / kg once a week (QW) for 2 weeks. Due to blood volume constraints, animals did not have blood samples taken at consecutive time points after multiple doses of the drug. Instead, all animals were administered a total of at least 4 doses of HIS-CGL-ILMDRGVS-PEG, and blood was collected 24, 48, 72, or 96 hours after 8, 6, 4, or 8 administrations for the BIW group and 5, 4, 3, or 2 administrations for the QW group, respectively. The starting levels of tHcy after washout were approximately 220 μM, and the greatest decrease was observed at 24 hours after administration in both groups (a decrease to approximately 50 μM). All repeat-dose groups maintained tHcy levels below 100 μM until 48 hours, and the 12.5 mg / kg BIW group maintained them below 100 μM until 72 hours (Figure 13).

[0140] Example 4: Intravenous (IV) pharmacokinetics of CGL-ILMDRGVS-PEG in monkeys The PK of CGL-ILMDRGVS-PEG was determined in untreated male cynomolgus monkeys. The PK study was conducted in the form of having two parts: Phase 1 (single intravenous (IV) administration) and Phase 2 (repeated subcutaneous (SC) administration). In Phase 1, the monkeys were administered CGL-ILMDRGVS-PEG by intravenous (IV) or subcutaneous (SC) injection on Day 1. For the same animals that received the first SC injection, a washout period of at least 14 days was set between the administrations of each phase, and then they were advanced to Phase 2. Following a washout period of approximately 14 days after the administration in Phase 1, each animal in the third group was administered CGL-ILMDRGVS-PEG at an appropriate dose level once a week (QW) for 3 consecutive weeks by SC injection (Table 4). In addition, each animal received SC administration of the vehicle control at the same volume and frequency at a location separate from the test substance. The test design is summarized in Table 4.

Table 4

[0141] The animals in Groups 1 and 2 received a single IV dose of CGL-ILMDRGVS-PEG. The animals in Group 3 (Phase 1) received a single SC dose of CGL-ILMDRGVS-PEG. A single IV dose of CGL-ILMDRGVS-PEG was administered to male monkeys in Groups 1 and 2 at 2 and 8 mg / kg.

[0142] IV exposure (C max , AUC 0-∞ ) increased approximately proportionally to the dose. The mean (±SD) C max was 72.3 ± 8.03 μg / mL at 2 mg / kg and 257 ± 18.7 μg / mL at 8 mg / kg. The mean (±SD) AUC 0-∞ was 7050 ± 419 and 24100 ± 4090 hr*μg / mL, respectively. Therefore, for a 4-fold change in dose, there were increases of approximately 3.6-fold and approximately 3.4-fold in the mean C max and AUC0-∞. The median of T max was observed at 0.5 hour after administration (Table 5 and Figures 14A, B).

Table 5

[0143] The circulating concentration of CGL-ILMDRGVS-PEG was higher at 8 mg / kg and measurable for a longer time compared to the circulating concentration at 2 mg / kg (Figure 14). The average clearance (CL) was slightly lower at the low dose, but the ranges (mean ± SD) overlapped (Table 5). The average (±SD) CL was 0.285 ± 0.0165 mL / hr / kg at 2 mg / kg and 0.339 ± 0.0629 mL / hr / kg at 8 mg / kg. The observed volume of distribution (V ss ) was almost equivalent to the monkey serum volume (45 mL / kg), but the average estimated value tended to be slightly lower. The average V ss (±SD) was 36.3 ± 4.40 mL / kg at 2 mg / kg and 41.6 ± 9.85 mL / kg at 8 mg / kg. The obtained average half-life (T 1 / 2 ) was in the range of 88.4 - 93.6 hours (about 3.7 - 3.9 days).

[0144] The increase in IV exposure (C max , AUC 0-∞ ) was approximately proportional to the dose (Table 5). The average (±SD) C max was 72.3 ± 8.03 μg / mL at 2 mg / kg and 257 ± 18.7 μg / mL at 8 mg / kg. The average (±SD) AUC 0-∞ was 7050 ± 419 and 24100 ± 4090 hr*μg / mL, respectively. Therefore, for a four-fold change in dose, there were approximately 3.6-fold and 3.4-fold increases in average C max and AUC 0-∞ , respectively. The T max median was observed at 0.5 hours after dosing.

[0145] Example 5: Pharmacokinetics of Subcutaneous (SC) CGL-ILMDRGVS-PEG in Monkeys Table 6 summarizes the average PK parameters in male monkeys after repeated SC dosing of CGL-ILMDRGVS-PEG on Day 1 and Day 15. The average serum concentration versus time profile is shown in Figure 15. [Table 6]

[0146] Male monkeys were repeatedly injected subcutaneously with CGL-ILMDRGVS-PEG at 8 (first dose) and 2 (second dose) mg / kg twice, with a 2-week washout period between administrations. The mean circulating concentration of CGL-ILMDRGVS-PEG was higher and measurable for a longer time at 8 mg / kg compared to the circulating concentration at 2 mg / kg (Figure 15). Thus, subcutaneous exposure (C max , AUC 0-∞ ) was higher at 8 mg / kg compared to 2 mg / kg (Table 6). The mean (±SD) C max was 106 ± 17.7 μg / mL at 8 mg / kg and 34.9 (N = 2) μg / mL at 2 mg / kg. The mean (±SD) AUC 0-∞ was 18,300 ± 1520 and 4710 (N = 1) hr*μg / mL, respectively.

[0147] T max he median was 48 hours at the high 8 mg / kg dose, which was slower, compared to 24 hours at 2 mg / kg (Table 6). The estimated mean bioavailability at 8 mg / kg was moderate at 75.8%. The estimated mean extravascular CL (CL / F) was slightly higher at 8 mg / kg than the IV CL estimate (e.g., 0.440 ± 0.0384 vs. 0.339 ± 0.0629 mL / kg / hr; Tables 5 and 6), indicating consistent incomplete absorption.

[0148] Example 6: Comparison of Pharmacodynamics of IV and SC The mean concentration-time profiles of plasma tHcy levels after IV and SC administration of CGL-ILMDRGVS-PEG are shown in Figures 16 and 17, respectively.

[0149] After IV administration of CGL-ILMDRGVS-PEG, the maximum decrease in plasma tHcy levels was observed at 4 - 8 hours after dosing, and further average decreases were observed at a high IV dosing level of 8 mg / kg (Figure 16). Five minutes after dosing (2 and 8 mg / kg respectively), there were initial decreases of approximately 39% and approximately 72% in plasma tHcy levels, after which they gradually decreased to the maximum decrease observed. At 8 hours after dosing, the average tHcy levels in the 2 and 8 mg / kg dosing groups were 47.8% and 18.1% of the average baseline levels respectively (corresponding to a 52.2% and 81.9% decrease), which corresponded to average CGL-ILMDRGVS-PEG concentrations of 58.6 and 194 μg / mL or more respectively. Recovery to baseline tHcy levels was almost complete (about 91%) by 240 hours in the 2 mg / kg intravenous dosing group, and about 84% was complete by 336 hours (2 weeks) in the 8 mg / kg intravenous dosing group.

[0150] After SC administration of CGL-ILMDRGVS-PEG, at the 8 mg / kg dose level, the maximum decrease in plasma tHcy levels was observed at 24 - 48 hours after dosing, and at the 2 mg / kg dose level at 8 - 24 hours after dosing (Figure 17). In contrast to IV administration, after dosing, a steady decrease in tHcy levels was seen until the maximum decrease was achieved. At the maximum decrease, the average tHcy levels were 75.5% and 42.0% of the average baseline levels for the 2 and 8 mg / kg dose levels respectively (corresponding to a 24.5% and 58.0% decrease), and note that 2 mg / kg of CGL-ILMDRGVS-PEG was administered 2 weeks after 8 mg / kg of CGL-ILMDRGVS-PEG (in the same animals). This corresponded to average CGL-ILMDRGVS-PEG concentrations of 34.9 and 98.7 μg / mL respectively, and note that there was significant inter-animal PK variability at 2 mg / kg. Recovery to baseline tHcy levels was complete (102%) by 336 hours or on day 15 of dosing (2 weeks) for the 8 mg / kg / day 1 dose. After the 2 mg / kg dose on day 15, recovery appeared to be complete by 168 hours after dosing (102%).

[0151] Example 7: Clinical Development Plan: First-in-Human Phase 1 / 2 Multiple Ascending Dose Trial The purpose of the Phase 1 / 2 multiple ascending dose trial is to examine the safety, pharmacokinetics, and pharmacodynamics of CGL-ILMDRGVS-PEG in patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. In this first-in-human study, randomization or blinding will not be performed, and the study will be non-blinded. The primary objectives, secondary objectives, and exploratory objectives are shown in Table 7 together with the corresponding endpoints. [Table 7]

[0152] Subjects can be included in the study if all of the following criteria apply: (i) diagnosis of homocystinuria due to CBS deficiency, (ii) ability to provide a signed informed consent / assent, including compliance with the requirements and restrictions listed in the informed consent form (ICF), and in this protocol, (iii) be over 12 years old at the time of signing the informed consent / assent, (iv) have plasma total homocysteine (tHcy) > 80 μM at least once at screening visit, (v) female subjects of childbearing potential must have a negative serum pregnancy test during the screening period before the first dose of the investigational drug and a negative urine pregnancy test before dosing on the first day of treatment, (vi) if the subject (male or female) is sexually active, they must agree to use a pregnancy prevention method that is either unable to cause pregnancy or is highly effective as defined in the full protocol, (vii) subjects receiving pyridoxine and / or betaine must have been on a stable dose of the drug for at least 6 weeks before the first dose of the investigational drug and intend to continue and be able to maintain a stable dose throughout the study period.

[0153] Subjects will be excluded from the study if any of the following criteria apply: (i) in the opinion of the responsible investigator, compliance with the study or interpretation of data (e.g., severe intellectual impairment preventing completion of required study evaluations), (ii) currently participating in another study or having received a dose of investigational drug within 30 days or the longer of five half-lives prior to the first dose of investigational drug in this study, (iii) having had surgery requiring general anesthesia within 8 weeks prior to the first administration of the investigational drug, (iv) having an infection requiring anti-infective therapy less than 2 weeks prior to the first dose of the investigational drug in this study (acceptable if anti-infective therapy was completed more than 2 weeks prior to the first dose of the investigational drug), (v) pregnancy or lactation, (vi) female subjects using estrogen-containing contraception or with a potential for pregnancy during the study, and (vii) a history of hypersensitivity to polyethylene glycol (PEG) such that, in the judgment of the investigator, the subject is placed at an unacceptable risk of adverse events (AE).

[0154] For cohort enrollment, each cohort will start with the administration to the sentinel subject. If CGL-ILMDRGVS-PEG is considered safe and has an acceptable tolerance profile at least 48 hours after monitoring following the administration to the sentinel subject, subsequent subjects can then be dosed.

[0155] Part 1: IV Administration Four subjects in Cohort 1 will receive an intravenous (IV) infusion of a dose of 0.15 mg / kg of CGL-ILMDRGVS-PEG over approximately 30 minutes. Across all cohorts, doses will be administered to subjects once weekly (QW) for up to 4 doses. After the 4 doses of CGL-ILMDRGVS-PEG have been administered to each of the first 2 subjects in Part 1, review the safety data, as well as all available PK and PD data. If the stopping rules are not met, transition to Part 2 will occur.

[0156] Part 2: SC Administration The subjects in Cohort 2 receive administration of CGL-ILMDRGVS-PEG at a dose of 0.15 mg / kg by subcutaneous (SC) injection. If it is assumed that the stopping rules are not met, the subjects in the second cohort receive a dose of 0.45 mg / kg, and the subjects in the third cohort receive a dose of 1.0 mg / kg. In all cohorts, a total of up to 4 doses administered QW are given to the subjects. If additional cohorts are added, the dose is determined based on data from the previous 3 cohorts, and after review of all safety, PK, and PD data from all planned cohorts, the dose can be up to 2 times the highest dose tested, and all of the following apply: (i) the safety stopping rules for dose escalation and trial termination are not met, and (ii) plasma tHcy (highest planned dose) taken 168 hours after dose 4 in one or more subjects in SC Cohort 3 (highest planned dose) is greater than 4 μM (lower limit of normal [LLN]).

[0157] The stopping rules and exceptions for the trial are described in Table 8.

Table 8

[0158] A schematic diagram of the human first-in-human Phase 1 / 2 multiple ascending dose trial to test CGL-ILMDRGVS-PEG is shown in Figure 3.

[0159] Throughout this specification, references to "one embodiment", "certain embodiments", "various embodiments", "one or more embodiments", or "an embodiment" mean that the particular features, structures, materials, or characteristics described in connection with the embodiment are included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one or more embodiments", "in certain embodiments", "in various embodiments", "in one embodiment", or "in an embodiment" at various places throughout this specification are not necessarily referring to the same embodiment of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0160] Although the disclosure of this specification is described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from its spirit and scope. Accordingly, the present disclosure is intended to cover modifications and variations that are within the scope of the appended claims and their equivalents.

Claims

**Claim 1** A method of treating a subject having or at risk of developing homocystinuria or hyperhomocysteinemia, comprising: administering a formulation comprising a modified human cystathionine-gamma-lyase (CGL) enzyme having at least the following substitutions relative to the native human CGL amino acid sequence (SEQ ID NO: 1): isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353, in a therapeutically effective amount; wherein the formulation is administered at a frequency of 1 dose per day to 1 dose per month, at an enzyme dose of about 0.05 mg / kg to about 4 mg / kg. **Claim 2** The method of claim 1, wherein the enzyme is conjugated with one or more PEG units. **Claim 3** The method of claim 1 or 2, wherein the formulation further comprises a pharmaceutically acceptable carrier. **Claim 4** The method of any one of claims 1 to 3, wherein the formulation is a 2 mL liquid supplied in a 5 mL vial. **Claim 5** The method of any one of claims 1 to 4, wherein the formulation comprises an enzyme concentration of about 1 mg / mL to about 50 mg / mL. **Claim 6** The method of any one of claims 1 to 5, wherein the formulation comprises an enzyme concentration of about 1 to about 20 mg / mL. **Claim 7** The method of any one of claims 1 to 6, wherein the formulation is administered intravenously or subcutaneously. **Claim 8** The method of any one of claims 1 to 7, wherein the formulation is administered at a dose of 1 dose per week. **Claim 9** The method of any one of claims 1 to 8, wherein the formulation is administered intravenously for 4 weeks and then subcutaneously in subsequent weeks. **Claim 10** The method of any one of claims 1 to 9, wherein the formulation is administered to the subject at a dose of about 0.1 mg / kg to about 1 mg / kg. **Claim 11** The method of any one of claims 1 to 10, wherein the formulation is administered once a week at a dose of about 0.1 mg / kg to about 1 mg / kg. **Claim 12** The method of any one of claims 1 to 11, wherein the formulation is administered to the subject at a dose of about 0.15 mg / kg, about 0.45 mg / kg, or about 1 mg / kg. **Claim 13** The method of any one of claims 1 to 12, wherein the formulation is diluted in physiological saline before being administered intravenously to the subject. **Claim 14** The method according to any one of claims 1 to 13, wherein the subject is a human patient.

15. The method according to any one of claims 1 to 14, wherein the subject has a total plasma homocysteine level of more than 80 μM before starting therapy by administering the formulation.

16. The method according to any one of claims 1 to 15, wherein the subject is at least 12 years old.

17. The method according to any one of claims 1 to 16, wherein the subject is maintained on an individualized diet.

18. The method according to any one of claims 1 to 17, wherein the subject is maintained on a methionine-restricted diet.

19. The method according to any one of claims 1 to 18, wherein the method reduces the total plasma homocysteine level.

20. The method according to any one of claims 1 to 19, wherein the method reduces the total plasma homocysteine level to about 80 μM or less.

21. The method according to any one of claims 1 to 20, wherein the method reduces the total plasma homocysteine level to about 50 μM or less.

22. The method according to any one of claims 1 to 21, wherein the method reduces the total plasma homocysteine level to about 15 μM or less.

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