Prokaryotic phenylalanine ammonia-lyase compositions and methods for treating adolescent subjects - Patents.com

Abstract

A phenylalanine ammonia-lyase (PAL) variant having higher phenylalanine converting activity and / or reduced immunogenicity compared to wild-type phenylalanine ammonia-lyase (PAL) for therapeutic uses, including treatment of adolescent subjects with PKU.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 190,567, filed May 19, 2021, which is incorporated by reference in its entirety.

[0002] (2. Sequence Listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "11808-479-228_SEQ_LISTING.txt," created on May 15, 2022, and having a size of 20,195 bytes.

[0003] 3. FIELD OF DISCLOSURE The present disclosure relates to prokaryotic phenylalanine ammonia lyase (PAL) and compositions thereof, and the optimization of such compositions to improve the catalytic activity and / or stability of prokaryotic PAL while decreasing its immunogenicity and / or proteolytic susceptibility. Further, the present disclosure relates to the use of such optimized compositions of prokaryotic PAL to treat adolescent subjects. [Background technology]

[0004] (4. Background of the Disclosure) PAL has been reported in plants (Koukol et al., J. Biol. Chem. 236:2692-2698 (1961); Hanson et al., The Enzymes 7:75-166 (1972); Poppe et al., Curr. Org. Chem. 7:1297-1315 (2003)), some fungi (Rao et al., Can. J. Biochem. 4512:1863-1872 (1967); Abell et al., Methods Enzymol. 142:242-253 (1987)), and bacteria (Bezanson et al., Can. J. Microbiol. 16:147-151 (1970); Xiang et al., J. Biol. Chem. 277:32505-32509 (2002); It is a non-mammalian enzyme that is widely distributed in Escherichia coli (Hill et al., Chem. Commun. 1358-1359 (2003)) and can be recombinantly produced in Escherichia coli.

[0005] PAL from the cyanobacterial strain Anabaena variabilis (Av) has been cloned and expressed in bacteria and shown to exhibit PAL enzyme activity in vitro and in vivo (see, e.g., U.S. Pat. Nos. 7,531,341; 7,534,595; 7,537,923; and 7,560,263). Pegylated recombinant Anabaena variabilis PAL (rAvPAL-PEG) has also been made, in which the rAvPAL protein has been derivatized by covalent attachment of polyethylene glycol (PEG) to increase its half-life and optimize its pharmacokinetic profile and / or reduce its immunogenicity (Id.). Recently, rAvPAL-PEG has been approved as an injectable product for the treatment of phenylketonuria (PKU) in adult subjects. There remains a need for methods for using such therapeutic agents in adolescent subjects. Summary of the Invention

[0006] (5. Disclosure Overview) In one aspect, provided herein is a method for reducing blood phenylalanine concentrations in a subject, comprising administering to the subject a weekly dose of a formulation comprising an AvPAL variant, wherein the subject is between about 12 and about 18 years of age, and the weekly dose is administered for greater than about 50 weeks, and the AvPAL variant comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the weekly dose is administered for more than about 60 weeks, more than about 70 weeks, more than about 80 weeks, more than about 90 weeks, more than about 100 weeks, more than about 110 weeks, more than about 120 weeks, more than about 130 weeks, more than about 140 weeks, more than about 150 weeks, more than about 160 weeks, more than about 170 weeks, more than about 180 weeks, more than about 190 weeks, more than about 200 weeks, more than about 210 weeks, more than about 220 weeks, more than about 230 weeks, more than about 240 weeks, or more than about 250 weeks.

[0007] In some embodiments, the dosage is in the range of about 0.1 mg per week to about 1 mg per week. In some embodiments, the dosage is in the range of about 1 mg per week to about 2 mg per week. In some embodiments, the dosage is in the range of about 2 mg per week to about 10 mg per week. In some embodiments, the dosage is in the range of about 10 mg per week to about 20 mg per week. In some embodiments, the dosage is in the range of about 20 mg per week to about 40 mg per week. In some embodiments, the dosage is in the range of about 40 mg per week to about 70 mg per week. In some embodiments, the dosage is in the range of about 70 mg per week to about 140 mg per week. In some embodiments, the dosage is in the range of about 140 mg per week to about 280 mg per week. In some embodiments, the dosage is in the range of about 280 mg per week to about 420 mg per week. In some embodiments, the dosage ranges from about 420 mg per week to about 840 mg per week.

[0008] In some embodiments, the AvPAL variant is administered once a week. In some embodiments, the AvPAL variant is administered twice a week. In some embodiments, the AvPAL variant is administered four times per week. In some embodiments, the AvPAL variant is administered seven times per week. In some embodiments, the AvPAL variant is administered 14 times per week. In some embodiments, the AvPAL variant is administered daily.

[0009] In some embodiments, the methods provided herein comprise administering to the subject the AvPAL variant at an induction dosage ranging from about 0.1 mg per week to about 10 mg per week, followed by administering to the subject the AvPAL variant at a titration dosage ranging from about 1 mg per week to about 200 mg per week, followed by administering to the subject the AvPAL variant at a maintenance dosage ranging from about 20 mg per week to about 840 mg per week. In some embodiments, the induction dosage is about 2.5 mg per week. In some embodiments, the titration dosage is in the range of about 5 mg per week to about 70 mg per week. In some embodiments, the maintenance dosage is in the range of about 140 mg per week to about 420 mg per week. In some embodiments, the induction dose is administered for a period of about 2 weeks to about 6 weeks, the dose titration dose is administered for a period of about 3 weeks to about 8 weeks, and the maintenance dose is administered for a period of about 50 weeks to about 80 weeks. In some embodiments, the induction dose is administered for a period of about 4 weeks, the dose titration dose is administered for a period of about 5 weeks, and the maintenance dose is administered for a period of about 56 weeks to about 64 weeks. In some embodiments, the maintenance doses consist of a first maintenance dose of about 70 mg per week to about 280 mg per week, a second maintenance dose of about 140 mg per week to about 560 mg per week, and a third maintenance dose of about 210 mg per week to about 840 mg per week. In some embodiments, the first maintenance dosage is administered for a period of about 16 weeks to about 24 weeks, the second maintenance dosage is administered for a period of about 16 weeks, and the third maintenance dosage is administered for a period of about 24 weeks.

[0010] In some embodiments, after administration of the maintenance dosage, the method further comprises administering to the subject an extended dosage of the AvPAL variant in the range of about 20 mg per week to about 840 mg per week, hi some embodiments, the extended dosage is administered for a period of about 40 weeks to about 120 weeks.

[0011] In some embodiments, the induction dose is administered for a period of about 4 weeks, the titration dose is administered for a period of about 5 weeks, the maintenance dose is administered for a period of about 64 weeks, and the extension dose is administered for a period of about 80 weeks.

[0012] In some embodiments, the methods provided herein further comprise assessing said blood phenylalanine concentration prior to administering said loading dose.

[0013] In some embodiments, the method further comprises evaluating the blood phenylalanine concentration after administration of one or more induction doses, titration doses, maintenance doses, and / or extension doses.

[0014] In some embodiments, the method further comprises adjusting the dosage based on the blood phenylalanine concentration.In some embodiments, the dosage is adjusted to achieve a blood phenylalanine concentration of less than about 600 μM.In some embodiments, the dosage is adjusted to achieve a blood phenylalanine concentration of less than about 360 μM.In some embodiments, when blood phenylalanine concentration is greater than about 360 μM, the maintenance dosage is increased.

[0015] In some embodiments, the subject has phenylketonuria (PKU). In some embodiments, the subject is about 12 to about 15 years old. In some embodiments, the subject is about 16 to about 17 years old.

[0016] In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO: 4.

[0017] In some embodiments, the AvPAL variant is PEGylated. In some embodiments, the PEGylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of at least 1.6 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the PEGylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of at least 2.4 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the PEGylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of 3 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the PEGylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of 5 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the pegylation is accomplished by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of 6 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the pegylation is accomplished by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of 7 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the pegylation is accomplished by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of 8 polyethylene glycol per lysine residue of the AvPAL variant. In some embodiments, the pegylation is accomplished by reacting the AvPAL variant with NHS-activated polyethylene glycol in a ratio of 9 polyethylene glycol per lysine residue of the AvPAL variant.

[0018] In some embodiments, the AvPAL variant is administered in a formulation comprising a pharma- ceutically acceptable carrier that comprises a stabilizing agent. In some embodiments, the stabilizing agent is L-phenylalanine or a structural analog thereof. In some embodiments, the stabilizing agent is selected from the group consisting of L-phenylalanine, trans-cinnamic acid, and benzoic acid. In some embodiments, the stabilizing agent is trans-cinnamic acid. In some embodiments, the formulation further comprises sodium chloride, and tromethamine and tromethamine hydrochloride.

[0019] Other features and advantages of the present disclosure will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while representing preferred embodiments of the present disclosure, are given by way of example only, since various changes and modifications that do not depart from the spirit and scope of the present disclosure will be apparent to those skilled in the art from said detailed description. [Brief description of the drawings]

[0020] (6. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1] Figure 1A shows the percentage of subjects who met a blood Phe threshold of ≦600 μmol / L, Figure 1B shows the percentage of subjects who met a blood Phe threshold of ≦360 μmol / L, and Figure 1C shows the percentage of subjects who met a blood Phe threshold of ≦120 μmol / L.

[0021] [Diagram 2]FIG. 2A describes the study overview for Cohort A (persons aged 16-17 years): a single-arm, open-label study. FIG. 2B describes the study overview for Cohort B (persons aged 12-15 years): PEGValiase active treatment arm vs. diet-only control arm (treatment assignment randomized at enrollment). FIG. 2C shows a dosing schematic for the active drug arms of Cohort A and Cohort B. Nine Cohort B diet-only control subjects will follow the Part 1 assessment schedule (excluding PEGValiase dosing) from weeks 1 through 73, then repeat the Part 1 assessment schedule including PEGValiase dosing from weeks 74 through 146.

[0022] [Diagram 3]FIG. 3 shows the schedule of assessments for Part 1 (Cohort A and active drug arms of Cohort B). ACTH, adrenocorticotropic hormone; ADHD-RS IV, Attention Deficit Hyperactivity Disorder Rating Scale IV; BRIEF, Behavioral Rating Scale of Executive Function; ECG, electrocardiogram; eCRF, electronic case report form; HRV, hypersensitivity reaction visit; PK, pharmacokinetics; PKU, phenylketonuria; SC / ET, study completion / early discontinuation visit. a) All scheduled visits will be at the study clinic or by a home health nurse; non-visits will be by telephone. Assessments will be conducted pre-dose unless otherwise noted. b) After written informed consent, a screening assessment must be conducted within 28 days prior to Day 1 of Part 1. Subjects will be evaluated for blood Phe concentrations during screening / observation with two measurements 2-4 weeks apart. c) Self-administration of the first study drug dose will be performed at the study clinic; once eligibility is documented, the subject will self-administer the study drug daily. d) Must be completed prior to administration of the first dose of study drug. Additional observers identified after screening must be trained. e) PKU medical history will include peak blood Phe (and age), evaluation of metabolic control by the subject, and age at which the low Phe diet is discontinued. f) HIV, Hepatitis B and C screening will be performed. g) It is recommended that a urine sample be obtained as the first or second morning void. In case of elevated urinary protein based on test results, a repeat urine test should be performed. This repeat urine sample must be obtained on the first or second morning void in the morning to allow for accurate test results and may be performed by a home health nurse. h) Shall be performed by a local laboratory. i) Serum cortisol sample should be taken prior to morning study drug administration before 10:00 AM. For the subject's convenience, a home health nurse may collect the sample. If the two results are low and abnormal, the subject will be asked to perform additional sampling for plasma ACTH and a low-dose conventional ACTH stimulation test or local practice based ACTH testing method. j) It is recommended that a urine sample be obtained as a first or second morning void.Subjects with a confirmed urine / albumin creatinine ratio of ≥100 mg / g should see a nephrologist if the result was within normal range at baseline. Subjects with elevated results at baseline who subsequently see an increase from baseline of 100-200 mg / g should also see a nephrologist. k) Shall be performed locally (if applicable). If the urine pregnancy test is positive or equivocal, a serum pregnancy test (central laboratory) must be performed. l) Subjects should keep a home record of all food, beverages, special low protein foods, and medical foods consumed during the 3 consecutive days prior to the study visit. No changes in protein (dietary and medical foods) intake will be allowed in Part 1. A food diary will be provided at screening for use in reporting in Part 1, Week 1. m) Blood will be drawn prior to study drug administration (pre-dose). Focused PK sampling will be performed in all subjects at Week 73. Samples will be collected pre-dose, 2, 4, 8, 12, and 24 hours post-dose. The 24 hour sample will be collected prior to the next daily dose. n) Blood will be collected for plasma Phe analysis after fasting for 2.5-5 hours. o) Immunogenicity assays will include total anti-PEG-variase antibodies (TAb), anti-PAL IgG, anti-PAL IgM, anti-PEG IgM, anti-PEG IgG, and neutralizing antibodies (NAb). At the hypersensitivity reaction (HRV) visit, only anti-PEG-variase IgE will be assessed. p) Investigator assessment. q) Completed by caregiver / parent. r) Between scheduled visits, clinic personnel will contact the subject weekly to monitor if the subject is experiencing any issues with self-administration, and to ask and answer questions regarding AEs or concomitant medications. s) AEs and concomitant medications should be recorded whenever the subject is evaluated by study personnel. After signed informed consent and before the first dose of pegvaliase, only SAEs related to study procedures will be collected. After the first dose, all AEs and SAEs will be collected until 4 weeks after the last study drug dose or study completion visit / early discontinuation visit, whichever occurs last. If there is a skin reaction lasting ≥14 days, the skin reaction should be recorded on the eCRF.Subjects with injection site skin reactions lasting ≥14 days should see a dermatologist and undergo a skin biopsy (optional). It is recommended that photographs of the skin reaction be taken by the subject or institution to aid in evaluating the event; photographs may be collected by the sponsor. t) Subjects may be premedicated with H1 antagonists, H2 antagonists, and antipyretics (e.g., acetaminophen) approximately 2-3 hours prior to study drug, as determined by the investigator. If a nonsteroidal anti-inflammatory drug (NSAID) is administered as a premedication, it should be given with food. If non-ambulatory, subjects will be asked about self-administration of study drug.

[0023] [Figure 4]FIG. 4 illustrates the schedule of assessments for Part 2 (Cohort A and active drug arms of Cohort B). ACTH, adrenocorticotropic hormone; ADHD-RS IV, Attention Deficit Hyperactivity Disorder Rating Scale IV; BRIEF, Behavioral Rating Scale of Executive Function; ECG, electrocardiogram; eCRF, electronic case report form; HRV, hypersensitivity reaction visit; PK, pharmacokinetics; PKU, phenylketonuria; SC / ET, study completion / early discontinuation visit. a) All scheduled visits will be at the study clinic or by a home health nurse; non-visits will be by telephone. Assessments will be conducted pre-dose unless otherwise noted. b) The Week 73 visit marks both the end of the primary treatment phase (Part 1) and the beginning of the extension phase (Part 2). The pre-dose assessments that constitute the end of the primary treatment phase and the administration of study drug that constitutes the beginning of the extension phase are shown in the Week 73 column in both FIG. 3 and FIG. 4. c) Additional observers identified after screening must be trained. d) It is recommended that the urine sample be obtained as the first or second morning void. In case of elevated urinary protein based on the test results, a repeat urine test should be performed. This repeat urine sample must be obtained on the first or second morning void in the morning to allow for accurate test results and may be performed by a home health nurse. e) It shall be performed by a local laboratory. f) Serum cortisol sample should be taken before the morning administration of the study drug before 10:00 AM. For the convenience of the subject, the home health nurse may collect the sample. If the two results are low and abnormal, the subject will be asked to perform additional sampling for plasma ACTH and a low-dose conventional ACTH stimulation test or an ACTH test method based on the facility's practice. g) It is recommended that the urine sample be obtained as the first or second morning void. Subjects with a confirmed urine / albumin creatinine ratio of ≥ 100 mg / g should be referred to a nephrologist if the results were within the normal range at baseline. Subjects with elevated results at baseline who subsequently demonstrate an increase of 100-200 mg / g from baseline should also be referred to a nephrologist. h) Shall be performed locally (where applicable).If the urine pregnancy test is positive or equivocal, a serum pregnancy test (central laboratory) must be performed. i) Subjects should keep a home record of all foods, beverages, proprietary low protein foods, and medical foods consumed for 3 consecutive days prior to the study visit. No changes in protein (dietary and medical foods) intake will be permitted in Part 1. A food diary will be provided at screening for use in reporting in Part 1, Week 1. j) Blood will be collected prior to study drug administration (pre-dose). Focused PK sampling will be performed in all subjects at Week 73. Samples will be collected pre-dose, 2, 4, 8, 12, and 24 hours post-dose. The 24 hour sample will be collected prior to the next daily dose. k) Blood will be collected for plasma Phe analysis after a 2.5-5 hour fast. l) Immunogenicity assays include total anti-PEGvaliase antibodies (TAb), anti-PAL IgG, anti-PAL IgM, anti-PEG IgM, anti-PEG IgG, and neutralizing antibodies (NAb). At the hypersensitivity reaction (HRV) visit, only anti-PEGvaliase IgE is assessed. m) Investigator assessment. n) Completed by caregiver / parent. o) Between scheduled visits, clinic personnel will contact the subject weekly to monitor if the subject is having issues with self-administration, ask about AEs or concomitant medications, and answer questions. p) AEs and concomitant medications should be recorded whenever the subject is assessed by study personnel. All AEs and SAEs will be collected until 4 weeks after the last study drug dose or study completion visit / early discontinuation visit, whichever occurs last. If there is a skin reaction that persists for ≥14 days, the skin reaction should be recorded on the eCRF. Subjects with injection site skin reactions lasting ≥14 days should see a dermatologist and undergo a skin biopsy (optional). It is recommended that photographs of the skin reaction be taken by the subject or institution to aid in evaluating the event; photographs may be collected by the sponsor. q) Subjects may be premedicated with an H1 antagonist, an H2 antagonist, and an antipyretic (e.g., acetaminophen) approximately 2-3 hours prior to study drug, as determined by the investigator.If a nonsteroidal anti-inflammatory drug (NSAID) is administered as a premedication, it should be given with food. In the non-attendant cases, subjects will be asked about self-administration of the study drug.

[0024] [Diagram 5]Figure 5 illustrates the schedule of assessments for Part 1 (cohort B diet-only control arm). ACTH, adrenocorticotropic hormone; ADHD-RS IV, Attention Deficit Hyperactivity Disorder Rating Scale IV; BRIEF, Behavioral Rating Scale of Executive Function; ECG, electrocardiogram; eCRF, electronic case report form; HRV, hypersensitivity reaction visit; PK, pharmacokinetics; PKU, phenylketonuria; SC / ET, study completion / early discontinuation visit. a) All scheduled visits will be at the study clinic or by the home health nurse; non-visits will be by telephone. b) After written informed consent, a screening assessment must be performed within 28 days prior to Day 1 of Part 1. Subjects will be evaluated for blood Phe concentrations during screening / observation with two measurements 2-4 weeks apart. c) Self-administration of the first study drug dose will occur in clinic in Part 2; once eligibility is documented, subjects will self-administer study drug daily during Part 2. d) Must be completed prior to administration of first dose of study drug in Part 2. Additional observers identified after screening must be trained. e) PKU medical history includes peak blood Phe (and age), subject's assessment of metabolic control, and age at which low Phe diet was discontinued. f) HIV, Hepatitis B and C screening will be performed. g) Urine sample is recommended to be obtained as first or second morning void. In case of elevated urinary protein based on test results, a repeat urine test should be performed. This repeat urine sample must be obtained on first or second morning void in the morning to allow for accurate test results and may be performed by a home health nurse. h) Shall be performed by a local laboratory. i) Serum cortisol sample should be collected prior to morning study drug administration before 10:00 AM. For the subject's convenience, a home health nurse may collect the sample. If the two results are low and abnormal, the subject will be asked to perform additional sampling for plasma ACTH and a low-dose conventional ACTH stimulation test or local practice based ACTH testing method. j) It is recommended that a urine sample be obtained as a first or second morning void.Subjects with a confirmed urine / albumin creatinine ratio of ≥100 mg / g should see a nephrologist if the result was within normal range at baseline. Subjects with elevated results at baseline who subsequently see an increase of 100-200 mg / g from baseline should also see a nephrologist. k) Shall be performed on-site (if applicable). If the urine pregnancy test is positive or equivocal, a serum pregnancy test (central laboratory) must be performed. l) Subjects should record at home all foods, beverages, special low protein foods, and medical foods consumed during the 3 consecutive days prior to the study visit. No changes in protein (dietary and medical foods) intake will be allowed in Part 1. At screening, a food diary will be provided for use in reporting in Part 1, Week 1. m) Blood will be drawn for plasma Phe analysis after fasting for 2.5-5 hours. n) Investigator assessment. o) Completed by caregiver / parent. p) Between scheduled visits, clinic personnel will contact the subject weekly to inquire about and answer questions regarding AEs or concomitant medications. q) AEs and concomitant medications should be recorded whenever the subject is evaluated by the study personnel. After signed informed consent and prior to Day 1, only SAEs related to study procedures will be collected. Starting with the Day 1 visit, all AEs and SAEs will be collected until 4 weeks after the last study drug dose or study completion / early discontinuation visit, whichever occurs last. If there is a skin reaction that persists for ≥14 days, the skin reaction should be noted on the eCRF. Subjects with injection site skin reactions that persist for ≥14 days should see a dermatologist for a skin biopsy (optional). It is recommended that photographs of the skin reaction be taken by the subject or institution to help evaluate the event; photographs may be collected by the sponsor.

[0025] [Figure 6]FIG. 6 illustrates the schedule of assessments for Part 2 (treatment with pegvaliase versus diet-only control arm of Cohort B). ACTH, adrenocorticotropic hormone; ADHD-RS IV, Attention Deficit Hyperactivity Disorder Rating Scale IV; BRIEF, Behavioral Rating Scale of Executive Function; ECG, electrocardiogram; eCRF, electronic case report form; HRV, hypersensitivity reaction visit; PK, pharmacokinetics; PKU, phenylketonuria; SC / ET, study completion / early discontinuation visit. All scheduled visits are at the study clinic or by a home health nurse; non-visits are by telephone. a) Assessments are conducted pre-dose unless otherwise noted. b) For Cohort B control subjects, the week 73 visit marks both the end of Part 1, where the subject's PKU is treated with diet alone, and the beginning of Part 2, where the subject is given pegvaliase. The week 73 pre-dose assessments that constitute the end of Part 1 are shown in the week 73 column of FIG. 5. The same assessments used as baseline assessments for Part 2 are shown in the Week 73 column of Figure 6 along with the administration of study drug that will begin Part 2. c) Self-administration of the first study drug dose will occur at the study clinic; once eligibility is documented, the subject will self-administer study drug daily. d) Must be completed prior to administration of the first dose of study drug. Additional observers identified after screening must be trained. e) It is recommended that a urine sample be obtained as the first or second morning void. In case of elevated urinary protein based on test results, a repeat urine test should be performed. This repeat urine sample must be obtained on the first or second morning void in the morning to allow for accurate test results and may be performed by a home health nurse. f) Shall be performed by a local laboratory. g) Serum cortisol sample should be collected prior to the morning study drug administration before 10:00 AM. For the subject's convenience, a home health nurse may collect the sample. If the two results are low and abnormal, the subject will be asked to perform additional sampling for plasma ACTH and a low-dose conventional ACTH stimulation test or local practice based ACTH testing method. h) It is recommended that a urine sample be obtained as a first or second morning void.Subjects with a confirmed urine / albumin creatinine ratio of ≥100 mg / g should see a nephrologist if the result was within normal range at baseline. Subjects with elevated results at baseline who subsequently see an increase of 100-200 mg / g from baseline should also see a nephrologist. i) Shall be performed locally (if applicable). If the urine pregnancy test is positive or equivocal, a serum pregnancy test (central laboratory) must be performed. j) Subjects should keep a home record of all foods, beverages, special low protein foods, and medical foods consumed during the 3 consecutive days prior to the study visit. No changes in protein (dietary and medical foods) intake will be allowed in Part 1. A food diary will be provided at screening for use in reporting in Part 1, Week 1. k) Blood will be drawn prior to study drug administration (pre-dose). Focused PK sampling will be performed in all subjects at Week 73. Samples will be taken pre-dose, 2, 4, 8, 12, and 24 hours post-dose. The 24 hour sample will be taken before the next daily dose. l) Blood will be taken for plasma Phe analysis after fasting for 2.5-5 hours. m) Immunogenicity assays will include total anti-PEG-variase antibodies (TAb), anti-PAL IgG, anti-PAL IgM, anti-PEG IgM, anti-PEG IgG, and neutralizing antibodies (NAb). At the hypersensitivity reaction (HRV) visit, only anti-PEG-variase IgE will be assessed. n) Investigator assessment. o) Completed by caregiver / parent. p) Between scheduled visits, clinic staff will contact the subject weekly to monitor if the subject is having problems with self-administration, ask about AEs or concomitant medications, and answer questions. q) AEs and concomitant medications should be recorded whenever the subject is assessed by study personnel. All AEs and SAEs will be collected until 4 weeks after the last study drug dose or study completion visit / early discontinuation visit, whichever occurs last. If there is a skin reaction lasting ≥ 14 days, the skin reaction should be recorded on the eCRF. Subjects with injection site skin reactions lasting ≥ 14 days should see a dermatologist and have a skin biopsy (optional).It is recommended that photographs of the skin reaction be taken by the subject or institution to aid in evaluating events; photographs may be collected by the sponsor. r) Subjects may be premedicated with an H1 antagonist, an H2 antagonist, and an antipyretic (e.g., acetaminophen) approximately 2-3 hours prior to study drug, as determined by the investigator. If a nonsteroidal anti-inflammatory drug (NSAID) is administered as a premedication, it should be given with food. In non-ambulatory cases, subjects will be asked about self-administration of study drug.

[0026] [Figure 7] Figure 7A shows the sequence of wild-type AvPAL (SEQ ID NO: 1), and Figure 7B shows the sequences of three AvPAL variants (SEQ ID NOs: 2 to 4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] (7. Detailed Description of Disclosure) The present disclosure is based, in part, on the favorable effects of treating adolescent subjects (e.g., subjects ages 12-17) suffering from PKU with rAvPAL according to the dosing regimens provided herein. Accordingly, in one aspect, provided herein is a method for treating an adolescent subject suffering from PKU comprising administering rAvPAL as provided herein according to the dosing regimens described herein, e.g., in the Examples section below.

[0028] (definition) Unless otherwise noted, the following terms used in this application, including the specification and claims, have the definitions set forth below. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly requires otherwise. Definitions of standard chemical terms can be found in reference texts such as Carey and Sundberg, Advanced Organic Chemistry, Third Edition, Vols. A and B (Plenum Press, New York 1992). The practice of the present disclosure employs, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectrometry, preparative and analytical methods of chromatography, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. See, e.g., TE Creighton, Proteins: Structures and Molecular Properties, (WH Freeman and Company, 1993); AL Lehninger, Biochemistry, (Worth Publishers, 4th ed., 2004); Sambrook et al., Molecular Cloning: A Laboratory Manual, (2nd ed., 1989); Methods In Enzymology, (S. Colowick and N. Kaplan, eds., Academic Press); Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0029] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0030] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include those that contain non-natural bases, nucleotides linked to other nucleotides by bonds other than naturally occurring phosphodiester bonds, or bases linked via bonds other than phosphodiester bonds. Nucleotide analogs thus include, but are not limited to, for example, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, generally not exceeding about 50 nucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood to also include RNA sequences in which "T" is replaced by "U" (i.e., A, U, G, C).

[0031] "cDNA" means a DNA complementary to or identical to an mRNA in either single-stranded or double-stranded form.

[0032] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to the nascent RNA transcript is referred to as the transcription direction. The DNA strand that has the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand that has the same sequence as the mRNA transcribed from that DNA and is located 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand that has the same sequence as the RNA and is 3' to the 3' end of the coding RNA transcript is referred to as the "downstream sequence".

[0033] "Complementary" refers to the topological compatibility or matching together of the interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and the properties of the contacting surfaces are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, a polynucleotide having the sequence 5'-TATAC-3' is complementary to a polynucleotide having the sequence 5'-GTATA-3'.

[0034] A nucleotide sequence is "substantially complementary" to a reference nucleotide sequence if the sequence complementary to the subject nucleotide sequence is substantially identical to the reference nucleotide sequence.

[0035] "Encoding" refers to the inherent property of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA made from the gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually shown in a sequence listing) and the non-coding strand (used as a template for transcription) of a gene or cDNA can be said to code for the protein or other product of the gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can include introns.

[0036] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally linked together. The amplified or constructed recombinant polynucleotide can be included in an appropriate vector, which can be used to transform an appropriate host cell. A host cell containing a recombinant polynucleotide is called a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide." A recombinant polynucleotide may also serve a non-coding function (e.g., promoter, origin of replication, ribosome binding site, etc.).

[0037] "Expression vector" means a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

[0038] "Amplification" means any means by which a polynucleotide sequence is copied and thus increased into a larger number of polynucleotide molecules, for example, by reverse transcription, polymerase chain reaction, and ligase chain reaction.

[0039] "Primer" refers to a polynucleotide that can specifically hybridize to a designated polynucleotide template to provide a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that induce synthesis, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization, such as DNA polymerase. Primers are usually single-stranded, but can also be double-stranded. Primers are usually deoxyribonucleic acids, but a variety of synthetic and natural primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize and serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such cases, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled, for example, with chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

[0040] "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-natural analogues thereof linked through peptide bonds, related naturally occurring structural variants, and synthetic non-natural analogues thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide.

[0041] Conventional notation is used herein to depict polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

[0042] "Conservative substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid. Each of the following six groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Amino acids can also be classified as follows: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basic: Asn, Gln, His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0043] The term "identical" or percent "identity," in the context of two or more polynucleotide or polypeptide sequences, means two or more sequences or subsequences that, when compared and aligned for best correspondence, have the same or a certain percentage of nucleotides or amino acid residues that are identical, as determined using a sequence comparison algorithm described in U.S. Pat. No. 7,553,653, which is incorporated herein by reference in its entirety, or by visual inspection.

[0044] In the context of two nucleic acids or polypeptides, the phrases "substantially homologous" or "substantially identical" generally refer to two or more sequences or subsequences that, when compared and aligned for best fit, have at least 40%, 60%, 80%, 90%, 95%, 98% nucleotide or amino acid residue identity as measured using one of the following sequence comparison algorithms or by visual inspection. Substantial identity may exist over a region of the sequence that is at least about 50 residues in length, for example, over a region of at least about 100 residues, or over a region of at least about 150 residues. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the comparison biopolymers.

[0045] "Substantially pure" or "isolated" means that the species of interest is the predominant species present (i.e., present on a molar basis in greater abundance than any other individual macromolecular species in the composition) and that the substantially purified fraction is a composition in which the species of interest constitutes at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80%-90% or more of the macromolecular species present in the composition are the purified species of interest. When a composition consists essentially of a single macromolecular species, the species of interest has been purified to essential homogeneity (contaminants cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ionic species are not considered macromolecular species for purposes of this definition. In some embodiments, the prokaryotic PAL variant composition is substantially pure or isolated. In some embodiments, the prokaryotic PAL variant compositions are substantially pure or isolated with respect to the macromolecular starting materials used in their synthesis, hi some embodiments, the pharmaceutical compositions comprise a substantially purified or isolated prokaryotic PAL variant in admixture with one or more pharma- ceutically acceptable excipients.

[0046] "Natural" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring.

[0047] "Wild-type" (wt) is a term referring to the naturally occurring genetic form of an organism. Wild-type is distinguished from mutant forms (organisms having a genetic mutation).

[0048] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed within the definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, as used herein, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. Such polypeptides are sometimes referred to herein as "mutants." These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations occurring in the host that produces the protein or errors due to PCR amplification.

[0049] As used herein, a "variant," "analog," or "derivative" is a compound, e.g., a peptide, that has more than about 70% but less than 100% sequence similarity with a given compound, e.g., a peptide. Such variants, analogs, or derivatives can be composed of non-natural amino acid residues, including, by way of example and not limitation, homoarginine, ornithine, penicillamine, and norvaline, as well as natural amino acid residues. Such variants, analogs, or derivatives can also be composed of one or more D-amino acid residues and can include non-peptidic interlinkages between two or more amino acid residues.

[0050] As used herein, a "ratio" of a PAL polypeptide (e.g., AvPAL or a variant thereof) to a water-soluble polymer (e.g., polyethylene glycol or PEG) refers to the molar ratio of the PAL polypeptide to the water-soluble polymer under reaction conditions. For example, a ratio of about 1:3 for AvPAL and polyethylene glycol (1:3 AvPAL:PEG) means that the chemically modified PAL was produced under reaction conditions with about 1 mole of lysine residues on AvPAL per 3 moles of polyethylene glycol. Because the AvPAL monomer has 18 lysine residues, a ratio of about 1:3 AvPAL:PEG corresponds to 1 mole of AvPAL per 54 moles of PEG in the pegylation reaction.

[0051] As used herein, "treatment" or "treating" refers to prophylactic or therapeutic or diagnostic treatment. A "prophylactic" treatment is a treatment administered to a subject who shows no signs or only early signs of a disease or pathology, i.e., PKU, for the purpose of reducing the risk of developing the pathology. Prokaryotic PAL compositions, such as the formulations provided herein, can be administered as a prophylactic treatment to reduce the likelihood of developing the pathology, i.e., PKU, or to minimize the severity of the pathology if it does develop. A "therapeutic" treatment is a treatment administered to a subject who shows signs or symptoms of a pathology, i.e., PKU, for the purpose of reducing or eliminating those signs or symptoms. The signs or symptoms can be biochemical, cellular, histological, functional, subjective, or objective. A prokaryotic PAL composition can be administered as a therapeutic treatment for diagnosis. "Diagnostic" refers to identifying the presence or nature of a pathology, i.e., PKU. Each diagnostic method differs in specificity and selectivity. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0052] As used herein, the terms "prevent," "preventing," and "prevention" refer to the complete or partial inhibition of the development, recurrence, onset, or spread of a disease and / or its associated symptoms (e.g., a disease or its associated symptoms associated with elevated phenylalanine levels, such as PKU in a patient) resulting from the administration of a therapy or combination of therapies provided herein, e.g., AvPAL, an AvPAL variant, or any derivative thereof.

[0053] "Pharmaceutical composition" means a composition suitable for pharmaceutical use in subjects, including humans and mammals. A pharmaceutical composition comprises a pharmacologically effective amount of a prokaryotic PAL polypeptide and also comprises a pharma- ceutical acceptable carrier. A pharmaceutical composition encompasses compositions that contain the active ingredient(s) and the inactive ingredient(s) that make up the carrier, as well as any products that result directly or indirectly as a result of combination, complexation, or aggregation of any two or more of the ingredients, or as a result of dissociation of one or more of the ingredients, or as a result of any other type of reaction or interaction of one or more of the ingredients. Thus, a pharmaceutical composition encompasses any composition made by combining a prokaryotic PAL polypeptide as provided herein and a pharma- ceutical acceptable carrier.

[0054] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical excipients, vehicles, diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers, such as, but not limited to, phosphate buffered saline, 5% dextrose in water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing, Easton, 1995). The pharmaceutical carrier used may depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0055] "Pharmaceutically acceptable" or "pharmacologically acceptable" means material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0056] As used herein, the term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (such as chimpanzees), and other ape and monkey species; domestic animals such as cows, horses, sheep, goats, and pigs; farm animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or sex. As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), and most preferably a human. In some embodiments, the subject is a mammal, preferably a human, who has been administered a PAL enzyme, such as AvPAL, or a variant thereof (e.g., SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4 (FIG. 7B)) and / or any derivative thereof (e.g., pegylated PAL), and / or any pharmaceutical composition and / or any pharmaceutical composition manufactured by any of the methods disclosed herein. In some embodiments of the methods and kits provided herein, the patient has a disease or condition associated with elevated phenylalanine levels, such as HPA or PKU (e.g., classical PKU, severe PKU, moderate PKU, or any subpopulation thereof). In some embodiments, the patient is a patient (e.g., a PKU patient) who has received an EST (e.g., rAvPAL or rAvPAL-PEG) for elevated phenylalanine levels. In another embodiment of the methods provided herein, the patient is fed a low-protein or protein-modified diet or a low-phenylalanine or phenylalanine-modified diet in combination with a pharmaceutical composition disclosed herein, such that plasma phenylalanine is reduced, for example, by at least about 25%.For further information regarding management of patient populations with elevated phenylalanine levels (e.g., HPA and PKU) with PAL or PAL-PEG (e.g., AvPAL or rAvPAL-PEG, or any variant thereof), which can be used in connection with the methods and kits provided herein in certain embodiments, see, e.g., U.S. Pat. Nos. 7,531,341 and 7,534,595.

[0057] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or ameliorate a disease associated with elevated phenylalanine levels (e.g., PKU) (or symptoms associated therewith). In certain embodiments, the terms "therapies" and "therapy" refer to biologic, supportive, and / or other therapies useful in the prevention, management, treatment, and / or amelioration of a disease associated with elevated phenylalanine levels (e.g., PKU) known to those of skill in the art, including medical practitioners.

[0058] The term "tissue" as used herein refers to tissue obtained from a mammal, e.g., a human. For example, the tissue may be from a biopsy sample, surgically removed tissue, or a post-mortem harvest. Additionally, the tissue may be homogenized and extracted to isolate enzymes or antibodies from the tissue.

[0059] Methods for Treating Adolescent Subjects In one aspect, provided herein is a method for reducing blood phenylalanine levels in an adolescent subject, comprising administering to the adolescent subject a formulation comprising a weekly dose of a pegylated AvPAL variant. In some embodiments, the subject is about 12 years to about 18 years of age. In some embodiments, the subject is about 12 years to about 17 years of age. In some embodiments, the subject is about 12 years to about 16 years of age. In some embodiments, the subject is about 12 years to about 15 years of age. In some embodiments, the subject is about 15 years to about 18 years of age. In some embodiments, the subject is about 16 years to about 18 years of age. In some embodiments, the subject is about 17 years to about 18 years of age. In some embodiments, the subject is about 15 years to about 17 years of age. In some embodiments, the subject is about 16 years to about 17 years of age. In some embodiments, the subject is about 12 years of age. In some embodiments, the subject is about 13 years old. In some embodiments, the subject is about 14 years old. In some embodiments, the subject is about 15 years old. In some embodiments, the subject is about 16 years old. In some embodiments, the subject is about 17 years old. In some embodiments, the subject is about 18 years old.

[0060] In some embodiments, provided herein is a method for reducing blood phenylalanine levels in a subject, comprising administering to the subject a weekly dose of a formulation comprising an AvPAL variant, wherein the subject is about 12 to about 18 years old, and the weekly dose is administered for more than about 50 weeks. In some embodiments, the weekly dose is administered for more than about 60 weeks. In some embodiments, the weekly dose is administered for more than about 70 weeks. In some embodiments, the weekly dose is administered for more than about 80 weeks. In some embodiments, the weekly dose is administered for more than about 90 weeks. In some embodiments, the weekly dose is administered for more than about 100 weeks. In some embodiments, the weekly dose is administered for more than about 110 weeks. In some embodiments, the weekly dose is administered for more than about 120 weeks. In some embodiments, the weekly dose is administered for more than about 130 weeks. In some embodiments, the weekly dose is administered for more than about 140 weeks. In some embodiments, the weekly dose is administered for greater than about 150 weeks. In some embodiments, the weekly dose is administered for greater than about 160 weeks. In some embodiments, the weekly dose is administered for greater than about 170 weeks. In some embodiments, the weekly dose is administered for greater than about 180 weeks. In some embodiments, the weekly dose is administered for greater than about 190 weeks. In some embodiments, the weekly dose is administered for greater than about 200 weeks. In some embodiments, the weekly dose is administered for greater than about 210 weeks. In some embodiments, the weekly dose is administered for greater than about 220 weeks. In some embodiments, the weekly dose is administered for greater than about 230 weeks. In some embodiments, the weekly dose is administered for greater than about 240 weeks, or greater than about 250 weeks.

[0061] In some embodiments, the dosage is in the range of about 0.1 mg per week to about 1 mg per week. In some embodiments, the dosage is in the range of about 1 mg per week to about 2 mg per week. In some embodiments, the dosage is in the range of about 2 mg per week to about 10 mg per week. In some embodiments, the dosage is in the range of about 10 mg per week to about 20 mg per week. In some embodiments, the dosage is in the range of about 20 mg per week to about 40 mg per week. In some embodiments, the dosage is in the range of about 40 mg per week to about 70 mg per week. In some embodiments, the dosage is in the range of about 70 mg per week to about 140 mg per week. In some embodiments, the dosage is in the range of about 140 mg per week to about 280 mg per week. In some embodiments, the dosage is in the range of about 280 mg per week to about 420 mg per week. In some embodiments, the dosage ranges from about 420 mg per week to about 840 mg per week.

[0062] In some embodiments, the AvPAL variant is administered once a week. In some embodiments, the AvPAL variant is administered twice a week. In some embodiments, the AvPAL variant is administered four times per week. In some embodiments, the AvPAL variant is administered seven times per week. In some embodiments, the AvPAL variant is administered 14 times per week. In some embodiments, the AvPAL variant is administered daily.

[0063] In some embodiments, the methods provided herein comprise administering to the subject an induction dosage of the pegylated AvPAL variant, followed by administering to the subject a titration dosage of the pegylated AvPAL variant, followed by administering to the subject a maintenance dosage of the pegylated AvPAL variant. In some embodiments, the induction dosage is administered for 1-5 weeks, e.g., 1, 2, 3, 4, or 5 weeks. In some embodiments, the titration dosage is administered for 4-10 weeks, e.g., 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the maintenance dosage is administered for 50 weeks or more, e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 weeks, or more. In some embodiments, the maintenance dose is administered for 50 to 70 weeks, hi some embodiments, the maintenance dose is administered for more than 70 weeks.

[0064] In some embodiments, the methods provided herein comprise administering to the subject the pegylated AvPAL variant at an induction dosage ranging from about 0.1 mg per week to about 10 mg per week, followed by administering to the subject the pegylated AvPAL variant at a titration dosage ranging from about 1 mg per week to about 200 mg per week, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage ranging from about 20 mg per week to about 840 mg per week. In some embodiments, the induction dosage is administered for 1-5 weeks, e.g., 1, 2, 3, 4, or 5 weeks. In some embodiments, the titration dosage is administered for 4-10 weeks, e.g., 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the maintenance dosage is administered for 50 weeks or more, e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 weeks, or more. In some embodiments, the maintenance dosage is administered for 50-70 weeks. In some embodiments, the maintenance dosage is administered for more than 70 weeks.

[0065] In some embodiments, the methods provided herein comprise administering to the subject the pegylated AvPAL variant at an induction dosage in the range of about 2.5 mg per week, followed by administering to the subject the pegylated AvPAL variant at a titration dosage in the range of about 5 mg per week to about 70 mg per week, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage in the range of about 140 mg per week to about 420 mg per week. In some embodiments, the induction dosage is administered for 1-5 weeks, e.g., 1, 2, 3, 4, or 5 weeks. In some embodiments, the titration dosage is administered for 4-10 weeks, e.g., 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the maintenance dosage is administered for 50 weeks or more, e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 weeks, or more. In some embodiments, the maintenance dosage is administered for 50-70 weeks. In some embodiments, the maintenance dosage is administered for more than 70 weeks.

[0066] In some specific embodiments, the methods provided herein comprise administering to the subject the pegylated AvPAL variant at an induction dosage in the range of about 2.5 mg per week for 4 weeks, followed by administering to the subject the pegylated AvPAL variant at a titration dosage in the range of about 5 mg per week to about 70 mg per week for 5 weeks, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage in the range of about 140 mg per week to about 420 mg per week for 56 to 64 weeks.

[0067] In some more specific embodiments, the methods provided herein comprise administering to said subject said pegylated AvPAL variant according to the dosing regimen of Table 3.

[0068] The pegylated AvPAL variants are described in more detail in the following sections. In some particular embodiments, the pegylated AvPAL variants provided herein are composed of recombinant phenylalanine ammonia lyase (rAvPAL) conjugated to N-hydroxysuccinimide (NHS)-methoxypolyethylene glycol (PEG). rAvPAL is a homotetrameric protein with a molecular weight of 62 kD per monomer. In some embodiments, an average of nine 20 kD PEG molecules are covalently attached (or conjugated) to each monomer of rAvPAL to produce the pegylated AvPAL variants (rAvPAL-PEG). In some embodiments, the total molecular weight of the pegylated rAvPAL is approximately 1000 kD. In some embodiments, the amino acid sequence of the rAvPAL monomer is SEQ ID NO:4, with the serine residues at positions 503 and 565 underlined in the sequence: [ka]

[0069] The chemical structure of N-hydroxysuccinimide (NHS)-methoxypolyethylene glycol (PEG) is shown below: [ka] As follows.

[0070] (Phenylalanine ammonia-lyase (PAL) and its variants) As used herein, "bacterial PAL" and "prokaryotic PAL" are used interchangeably and refer to (1) bacterial PALs that are active against a variety of bacteria, including, but not limited to, Streptomyces maritimus, Nostoc punctiforme, Anabaena variabilis, Anacystis nidulans (Lofflehardt, Z. Naturforsch. 31(11-12):693-9 (1976)), Photorabdus luminescens TT01 (Williams et al., Microbiology 151 :2543-2550 (2005)), and Streptomyces verticillatus (Bezanson et al., Can. J. (2) fragments, mutants, variants, or analogs of such wild-type PAL enzymes that maintain similar (i.e., at least 50%) catalytic activity towards phenylalanine and that may exhibit, for example, increased catalytic activity, greater biochemical stability, increased half-life, and / or reduced immunogenicity; and (3) chemically modified versions of such wild-type PAL enzymes or their fragments, mutants, variants, or analogs that are linked to other chemical moieties that provide other advantageous effects, such as, but not limited to, improved half-life and / or reduced immunogenicity. For example, reference to prokaryotic PAL, and fragments, mutants, variants, analogs, or chemically modified versions thereof, and methods of making or using compositions of such enzymes for therapy, is intended to mean methods of making, using, or formulating all such wild-type prokaryotic PAL, or fragments, mutants, variants, analogs, or chemically modified versions thereof.

[0071] One embodiment is a prokaryotic PAL from Anabaena variabilis (SEQ ID NO:1) (see FIG. 7A) or a biologically active fragment, mutant, variant, or analog thereof.

[0072] The elucidation of a reliable three-dimensional structure or structural model for a particular macromolecule allows rational design to be a productive method for optimizing the particular structure and / or function of the macromolecule. Methods for using three-dimensional structures or structural models to optimize PAL enzymes are described in U.S. Patent No. 7,553,653, the entirety of which is incorporated herein by reference. High-resolution three-dimensional protein crystal structures of prokaryotic PAL can be used in methods involving protein engineering to improve the biochemical and biophysical properties of prokaryotic PAL and to increase the therapeutic efficacy of prokaryotic PAL in vivo. In some embodiments, provided herein are prokaryotic PAL variants with higher phenylalanine conversion activity and / or reduced immunogenicity compared to wild-type prokaryotic PAL. Also provided herein are prokaryotic PAL variants with higher biochemical stability and / or longer biochemical half-life compared to wild-type prokaryotic PAL.

[0073] Previous studies have demonstrated that PAL mutants (Schuster et al., FEBS Lett. 349(2):252-254 (1994); Schuster et al., Proc Natl Acad Sci USA 92(18):8433-8437 (1995); Langer et al., Biochemistry 36: 10867-10871 (1997); El-Batal et al., Acta Microbiol Pol. 49(1):51-61 (2000); Rother et al., Eur. J. Biochem. 269:3065-3075 (2002)) and HAL mutants (Taylor et al., J. Biol. Chem. 269(44):27473- 27477 (1994); Baedeker et al., Eur. J. Biochem. Modified forms of PAL have been described, such as in J. Immunol. 269(6): 1790-1797 (2002)).

[0074] The bioactive site of the wild-type PAL provided herein can be modified to optimize the kinetic properties of PAL. The Km, the concentration of substrate that gives half-maximal activity, is closely related to the therapeutic efficacy of PAL in maintaining Phe levels within the acceptable range, i.e., 120 μM to 240 μM. Km is the affinity of the enzyme for the substrate. By controlling the affinity, the efficacy of any enzyme for the substrate at various concentrations can be limited or controlled. For example, if the Km is 1000 μM (e.g., PAL from Rhodosporidium toruloides), the activity of the enzyme will decrease to about 12.5% ​​at a blood Phe level of 240 μM and decrease to about 3% at a blood Phe level of 60 μM. If the Km is 240 μM, the activity of the enzyme will decrease by about 50%> at a blood Phe level of 240 μM and by about 12% at a blood Phe level of 60 μM. If the Km is 120 μM, the activity of the enzyme will decrease by about 70%> at a blood Phe level of 240 μM and by about 35%> at a blood Phe level of 60 μM. Preferably, the goal of treatment will be to have an enzyme with sufficient activity to lower but maintain Phe within the optimal range of about 120 μM to about 240 μM. An enzyme with a high Km (i.e., 1000 μM) will lose activity rapidly as Phe levels fall within the normal range and will require the administration of highly concentrated or impractical large doses. On the other hand, an enzyme with a very low Km may rapidly deplete Phe levels, which may be fatal in cases of hyperphenylalaninemia, but may be useful in managing the disease or disorder.

[0075] In some embodiments, the biologically active modified PAL has a kcat of at least about 0.1 s-1 or greater than about 0.5 s-1. In another embodiment, the biologically active modified PAL has a kcat of at least about 0.2 s-1 or greater than about 1.0 s-1. In another embodiment, the biologically active modified PAL has a Km of about 10 μM to about 1000 μM. In another embodiment, the biologically active modified PAL has a Km of about 100 μM to about 1000 μM. In another embodiment, the biologically active modified PAL exhibits an enzymatic activity that is about 2-fold to about 1000-fold higher than that of the wild type. In another embodiment, the biologically active modified PAL exhibits an enzymatic activity that is about 10% to about 100% higher than that of the wild type. Such biologically active modified PAL proteins can be generated using methods well known in the art, for example, by site-directed mutagenesis.

[0076] Several strategies are currently used to reduce protein immunogenicity. In some embodiments, the modifications introduced to minimize immune response do not destroy the structure, function, or stability of macromolecules. Effective strategies used include increasing human sequence content (chimera and / or other humanization methods), improving solution properties, removing antibody epitopes, introducing chemical derivatization (such as PEGylation), and / or identifying and removing MHC agretopes.

[0077] Modification of antigenic surface protein regions reduces immunogenicity (Chirino et al., Drug Discov. Today 9(2): 82-90 (2004)). One method of improvement involves the generation of proteins of smaller size that retain catalytic activity (e.g., absorbance assays are used to measure activity). Protein engineering combined with ELISA screening may also be used to identify mutants with reduced immunoreactivity. Another method introduces point mutations for additional surface Lys sites for derivatization by pegylation, a method that has been shown to reduce immunogenicity with the test enzyme purine nucleoside phosphorylase (Hershfield et al., 1991, supra). Another route utilizes mutations of residues located in protein epitope regions to remove immunogenic sites (Yeung et al., J. Immunol. 172(11):6658-6665 (2004)). In an approach similar to antibody humanization, homologous loop regions and / or residues from a human antibody are substituted into the corresponding loop regions of a homologous protein.

[0078] Improving the solution properties of proteins can increase specific enzymatic activity and / or reduce immunogenicity. One solution property unique to bacterially expressed recombinant proteins is the formation of protein aggregates due to, for example, inter-chain disulfide bind formation, hydrophobic interactions, and / or divalent cations (Chi et al., Pharm. Res. 20(9): 1325-1336 (2003)). Aggregation of recombinantly expressed proteins can enhance immune responses (Hermeling et al., Pharm. Res. 21(6): 897-903 (2994); Schellekens, Nephrol. Dial. Transplant. 20(suppl 6):vi3-9 (2005)). One method of improvement involves replacing surface cysteine ​​residues with other amino acid residues (e.g., serine) to minimize the possibility of inter-chain disulfide bond formation. For example, replacement of two surface cysteine ​​residues with serine residues reduced aggregation of chorismate lyase with a minor effect on enzyme activity (Holden et al., Biochim. Biophys. Acta 1594(1): 160-167 (2002)).

[0079] Also provided herein are prokaryotic PAL variants with similar or higher phenylalanine conversion activity and / or reduced immunogenicity compared to wild-type PAL. Further provided herein are prokaryotic PAL variants that include one or more amino acid residues (e.g., cysteine) that are replaced with another amino acid residue (e.g., serine) to reduce protein aggregation, which may be associated with reduced enzymatic activity, increased immunogenicity, and / or other adverse effects in vivo, such as reduced bioavailability. In some embodiments, provided herein are pharmaceutical compositions in which one or more amino acid residues of a prokaryotic PAL variant are replaced with another amino acid. In some embodiments, the replacement increases phenylalanine conversion activity and / or reduces immunogenicity compared to wild-type PAL.

[0080] In certain embodiments of the method or use, the prokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) variant. In some embodiments, one or more amino acid residues of the AvPAL variant are replaced with another amino acid residue. In some embodiments, one or more cysteine ​​residues of the AvPAL variant are replaced with a serine residue. In some embodiments, the one or more cysteine ​​residues of the AvPAL variant that are replaced with one or more serine residues are selected from the group consisting of the cysteine ​​residues at positions 503 and 565. In a specific embodiment, the cysteine ​​residue at position 503 of the AvPAL variant is replaced with a serine residue (e.g., SEQ ID NO:2). In some embodiments, the cysteine ​​residue at position 565 of the AvPAL variant is replaced with a serine residue (e.g., SEQ ID NO:3). In certain embodiments, the cysteine ​​residues at positions 503 and 565 of the AvPAL variant are replaced with a serine residue (e.g., SEQ ID NO:4).

[0081] Prokaryotic PAL variants also include fusion proteins in which the PAL enzyme is fused to another heterologous polypeptide, such as a native or modified constant region of an immunoglobulin or a fragment thereof carrying a salvage epitope known in the art to increase half-life.

[0082] (PEGylated PAL) Macromolecule chemical modification can be performed in a non-specific manner (leading to a mixture of derivatized species) or in a site-specific manner (based on site-selective modification using wild-type macromolecule reactivity-directed derivatization and / or a combination of site-directed mutagenesis and chemical modification) or using expressed protein ligation methods (Hofmann et al., Curr. Opin. Biotechnol. 13(4):297-303 (2002)). In certain embodiments, chemical modification is used to reduce immunogenicity. Pegylation is a proven method to reduce the immunogenicity of proteins (Bhadra et al., Pharmazie 57(1):5-29 (2002)), but other chemical derivatization techniques are also possible, using glycosylation and modifications such as phosphorylation, amidation, carboxylation, acetylation, methylation, acid addition salts, amides, esters, and the generation of N-acyl derivatives (Davis, Science 303:480-482 (2004)).

[0083] A series of different PEGylation reactions on PAL using various ratios of PEG chemical reagent to PAL protein will result in PEG-PAL derivatives for each modification method. The optimal degree of PEGylation can be determined based on the residual activity obtained for each derivatized PAL species using absorbance assays combined with PAGE and native gel analysis or SE-HPLC with multi-angle light scattering (MALS) to determine the extent of PEG derivatization. After the initial range of optimal modification is determined, comparative kinetic analysis (including Vmax and Km determination, substrate binding constants, proteolytic stability, pH dependence of activity, temperature dependence of activity) and immunoreactivity of the optimal PEG-PAL species can be determined by ELISA, immunoprecipitation, and Western blot. Protein engineering can also be used to generate PAL variants that are most favorable for PEGylation using optimal derivatization conditions; minimizing the size of the PAL protein and modifying only the most antigenic regions of the PAL surface will simultaneously lower the cost of PEG modification while maintaining the maximum amount of enzymatic activity and the minimum amount of immunogenicity. Similarly, site-specific pegylation can be used to provide enzyme derivatives.

[0084] Other chemical modifications such as phosphorylation or other chemical modifications of Lys, Arg, and Cys residues can be used to mask immunogenic and / or proteolytically sensitive regions. Such chemical modifications include Bednarsaki's polymer addition method to improve PAL stability, reduce immunogenicity, and improve protease resistance, and Altus's cross-linking method is representative. Bednarsaki demonstrated that polymer addition improves the temperature stability of proteins (Wang et al., J. Am. Chem. Soc. 114(1):378-380 (1992)), and Altus found that glutaraldehyde cross-linking improves enzyme stability.

[0085] To determine whether the in vivo therapeutic half-life of a protein such as PAL would benefit from PEGylation, a variety of different PEG:PAL conjugates were synthesized, characterized in vitro, and tested in vivo for the reduction of L-Phe. A design strategy was employed that varied in polymer length, conformation, and degree of PEG conjugation to both optimize the potential effect of PEGylation and identify one or more sites of PEG attachment. In some embodiments, the method for preparing PEGylated PAL generally includes: (a) reacting PAL with polyethylene glycol under conditions where PAL binds to one or more PEG groups, and (b) obtaining a reaction product. Since the specific site of PAL modification can significantly alter the intrinsic activity of the conjugate, various types and amounts of PEG were investigated. The chemistry used for PEGylation of PAL was acylation of the primary amine of PAL with the NHS ester of methoxy-PEG (O-[(N-succinimidyloxycarbonyl)-methyl]-O'-methylpolyethylene glycol). Acylation with methoxy-PEG-NHS or methoxy-PEG-SPA results in an amide bond that removes the charge from the original primary amine.

[0086] The method provides a substantially homogenous mixture of polymer:protein conjugates. As used herein, "substantially homogenous" means that only polymer:protein conjugate molecules are found. The polymer:protein conjugates have biological activity, and the "substantially homogenous" PEGylated PAL preparations provided herein are sufficiently homogenous to exhibit the advantages of homogenous preparations, such as ease of clinical application in lot-to-lot pharmacokinetic predictability.

[0087] The polymer molecules envisioned for use in the PEGylation techniques described herein can be selected from water-soluble polymers or mixtures thereof. Water-soluble polymers can be selected from the group consisting of, for example, polyethylene glycol, monomethoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone), propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), HPMA, Fleximer™, and polyvinyl alcohol, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonate PEG, cellulose, or other carbohydrate-based polymers. The polymer selected should be water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be branched or unbranched. In some embodiments, for therapeutic use of the final product preparation, the polymer will be pharma- ceutically acceptable.

[0088] In some embodiments, the water-soluble polymer for use herein is polyethylene glycol, abbreviated as PEG. As used herein, polyethylene glycol is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono-(C1-C10)alkoxy- or aryloxy-polyethylene glycol.

[0089] The ratio of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the optimal ratio (in terms of efficiency of the reaction in terms of not having excess unreacted protein or polymer) will be determined by the molecular weight of the polyethylene glycol selected and based on the number of available reactive groups (usually ε-amino groups) present. In general, the higher the molecular weight of the polymer used, the fewer the number of polymer molecules that can be attached to the protein. Similarly, branching of the polymer can be taken into consideration when optimizing these parameters. In general, the higher the molecular weight (or the more branching), the higher the polymer:protein ratio. Several different linear PEG polymer lengths are contemplated, including but not limited to 5 kDa and 20 kDa, and two-arm branched PEG polymer conjugates are contemplated, including but not limited to 10 kDa and 40 kDa. In some embodiments, for the PEGylation reactions contemplated herein, the average molecular weight is about 2 kDa to about 100 kDa (the term "about" indicates + / - 1 kDa). In another embodiment, the average molecular weight is from about 5 kDa to about 40 kDa.

[0090] Examples 7-9 of commonly owned U.S. Pat. No. 7,531,341, the entire contents of which are incorporated herein by reference, show that ENU2 or BTBR enu2We describe the effect of PEGylated and non-PEGylated forms of lysine mutant R91K PAL (RtPAL), NpPAL, and AvPAL from Rhodosporidium toruloides on Phe levels in mice. This animal model is homozygous mutant at the PAH locus resulting in animals with severe hyperphenylalaninemia. The high plasma Phe levels make this a suitable model to evaluate the ability of PAL to lower plasma Phe. Administration of PEGylated forms of NpPAL and AvPAL resulted in greater reduction of Phe in ENU2 mice compared to non-PEGylated NpPAL and AvPAL, respectively. Such an effect was maintained over 10 weeks with weekly injections of NpPAL. These results indicate that PEGylation of PAL from the cyanobacteria Nostoc punctiforme and Anabaena variabilis is essential to reduce Phe levels in mice affected by PKU.

[0091] The effect of serine substitution of cysteine ​​residues in AvPAL polypeptide (e.g., those at positions 503 and 565) on Phe levels in ENU2 mice has also been shown. Administration of pegylated AvPAL double cysteine ​​mutant (positions 503 and 565) AvPAL_C565SC503S results in a reduction in plasma Phe that is comparable to that achieved with pegylated wild-type AvPAL. It has been shown that AvPAL_C565SC503S has in vivo PAL enzyme activity comparable to that of pegylated wild-type AvPAL, and has reduced immunogenicity compared to pegylated wild-type AvPAL.

[0092] Pegylated PAL variants with reduced immunogenicity are provided herein. One embodiment is a pegylated form of AvPAL variant with reduced immunogenicity. Certain embodiments contemplate AvPAL variants in which pegylation is achieved by reacting the AvPAL variant with a water-soluble polymer, e.g., polyethylene glycol (PEG). In some embodiments, pegylation is achieved by reacting the AvPAL variant with PEG once in a ratio of at least 1:1, at least 1:1.5, at least 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:6, at least 1:7, at least 1:8, at least 1:9, or at least 1:10 (PAL:PEG). In one embodiment, the PAL variant is an AvPAL variant and the pegylation is achieved using a PAL:PEG ratio of about 1:1 to about 1:20. In another embodiment, the PAL variant is an AvPAL variant and the pegylation is accomplished using a PAL:PEG ratio of about 1:3 to about 1:12. In yet another embodiment, the PAL variant is an AvPAL variant and the pegylation is accomplished using a PAL:PEG ratio of about 1:5 to about 1:10. In yet another embodiment, the PAL variant is an AvPAL variant and the pegylation is accomplished using a PAL:PEG ratio of about 1:9.

[0093] In certain embodiments, one or more lysine residues are introduced into and / or near the active site of a prokaryotic PAL variant to improve catalytic activity, reduce immunogenicity, and / or improve biochemical stability, in part by preventing possible PEGylation of other amino acid residues (e.g., tyrosine) at and / or near the active site of the enzyme, or by preventing possible PEGylation of lysine residues important for enzymatic activity. Without being bound by a particular theory, it is hypothesized that the tyrosine residue at and / or near the active site of prokaryotic PAL (i.e., position 78 or 314 in AvPAL) can be a site for PEGylation to reduce enzymatic activity. In some embodiments, one or more amino acid residues at and / or near the active site of prokaryotic PAL that are not required for enzymatic activity are replaced with lysine residues. In certain embodiments, the prokaryotic PAL is AvPAL. In one embodiment, the AvPAL tyrosine residue at position 78 or 314 is not available for PEGylation. Again, without being bound by theory, it is hypothesized that the lysine residue of prokaryotic PAL (i.e., position 419 of AvPAL) that is normally prevented from pegylation due to the pegylation of the adjacent lysine residue PAL (i.e., position 413 of AvPAL) can be a site for pegylation, which reduces substrate binding and / or catalytic activity. In some embodiments, one or more amino acid residues of prokaryotic PAL are replaced with lysine residues such that the lysine residue important for substrate binding and / or catalytic activity of the enzyme is not available for pegylation. In a specific embodiment, the prokaryotic PAL is AvPAL. In one embodiment, the AvPAL lysine residue at position 419 is not available for pegylation.

[0094] In some embodiments, the composition comprises a highly purified bacterially derived prokaryotic PAL variant, or a biologically active fragment, mutant, or analog thereof, alone or in combination with a medicament suitable carrier. In some embodiments, the preparation contains a prokaryotic PAL variant with a purity of greater than about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In another embodiment, the relative specific activity of the prokaryotic PAL variant is at least about 50% or greater than about 110% of the specific activity of wild-type prokaryotic PAL.

[0095] Such prokaryotic PAL variants can be isolated and purified according to methods known in the art, and are present in amounts that allow the prokaryotic PAL enzyme to be used therapeutically. In some embodiments, cDNA encoding the complete or wild-type prokaryotic PAL is used. However, in other embodiments, cDNA encoding a biologically active fragment, mutant, variant, or analog thereof can be used. Additionally provided herein are compositions of optimized prokaryotic PAL and / or chemically modified (e.g., pegylated) forms of PAL obtained by structure-based molecular engineering approaches. Certain embodiments contemplate optimal compositions of prokaryotic PAL with improved specific activity, improved stability, reduced immunogenicity, and / or proteolytic susceptibility suitable for therapeutic use. In some embodiments, the PAL is a pegylated form of Anabaena variabilis PAL with improved specific activity, improved stability, reduced immunogenicity, and / or proteolytic susceptibility.

[0096] In some embodiments, the pegylated prokaryotic PAL variant is an AvPAL variant in which the cysteine ​​residue at position 503 of AvPAL has been replaced with a serine residue (SEQ ID NO:2). In some embodiments, the pegylated prokaryotic PAL variant is an AvPAL variant in which the cysteine ​​residue at position 565 of AvPAL has been replaced with a serine residue (SEQ ID NO:3). In some embodiments, the pegylated prokaryotic PAL variant is an AvPAL variant in which the cysteine ​​residues at positions 503 and 565 of AvPAL have been replaced with serine residues (SEQ ID NO:4).

[0097] Prokaryotic PAL Compositions, Pharmaceutical Compositions, and Formulations The present disclosure contemplates pharmaceutical compositions comprising a therapeutically effective amount of the prokaryotic PAL compositions of the present disclosure together with one or more pharma- ceutically acceptable excipients, vehicles, diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such pharmaceutical compositions include diluents of various buffer contents (e.g., Tris-HCl, phosphate), pH, and ionic strength; surfactants and solubilizers (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol); see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.), pp. 1435-1712, which is incorporated herein by reference. An effective amount of active ingredient is a therapeutically, prophylactically, or diagnostically effective amount, which can be readily determined by one skilled in the art by taking into consideration factors such as weight, age, and goal of treatment.

[0098] The prokaryotic PAL pharmaceutical composition of the present disclosure may include a buffering agent that maintains the pH of the solution within a desired range. Suitable buffering agents include Tris-HCl, sodium acetate, sodium phosphate, and sodium citrate. Mixtures of these buffering agents may be used. The amount of buffering agent useful in the composition will depend primarily on the particular buffering agent used and the pH of the solution. For example, acetic acid is a more efficient buffering agent at pH 5 than at pH 6, and therefore less acetic acid may be used in a pH 5 solution than at pH 6. A more suitable buffering agent is Tris-HCl. A suitable pH range for the pharmaceutical composition of the present disclosure is about pH 6.0-8.5. A more suitable pH range for the pharmaceutical composition of the present disclosure is about pH 7.0-8.0. A most suitable pH range for the pharmaceutical composition of the present disclosure is about pH 7.0-7.6.

[0099] Additionally, the pharmaceutical composition of the present disclosure may include an isotonicity adjusting agent to make the solution isotonic and to enhance suitability for injection. A preferred agent is sodium chloride in the concentration range of 50-200 mM. A more preferred agent is sodium chloride in the concentration range of 100-150 mM. A most preferred agent is sodium chloride in the concentration range of 130-150 mM.

[0100] Pharmaceutically acceptable carriers or excipients may include stabilizers, which are molecules that stabilize the prokaryotic PAL compositions of the present disclosure. As used herein, the term "stabilize" is intended to include, but is not limited to, for example, extending the shelf life of the prokaryotic PAL enzyme, protecting the prokaryotic PAL enzyme from proteolytic digestion, maintaining the prokaryotic PAL enzyme in an active conformation, and preserving prokaryotic PAL enzyme activity upon storage at elevated temperatures.

[0101] Stabilizers of the present disclosure include L-phenylalanine (Phe) and its structural analogs, such as trans-cinnamic acid (t-CA), benzoic acid, tyrosine (Tyr). Loss of activity of plant PAL (PvPAL) from Phaseolus vulgaris has been shown upon removal of its substrate L-phenylalanine after affinity purification (Da Cunha et al., Eur. J. Biochem. 178:243-248 (1988)), and yeast PAL (RtPAL) from Rhodosporidium toruloides has been shown to be protected from protease inactivation by tyrosine (Wang et al., Mol. Genet. Metab. 86:134-140 (2005); Pilbak et al., FEBS J. 273:1004-1019 (2006)). As shown herein below, Phe and some of its structural analogs have the ability to stabilize PEG:PAL conjugates of prokaryotic PAL (AvPAL) from Anabaena variabilis (see Example 11). Without being bound to a particular theory, it is hypothesized that the prokaryotic PAL enzyme is more stable as an enzyme-substrate complex in which the bound substrate Phe is converted to the product t-CA or to a transition state analog of t-CA. t-CA remains bound to the center of the otherwise highly reactive active site (the MIO group), thereby stabilizing the PAL enzyme. Thus, the PAL enzyme substrate Phe, the product t-CA, or structural analogs thereof are stabilizers of the present disclosure.

[0102] The present disclosure contemplates a pharmaceutical composition comprising a prokaryotic PAL variant and a pharma- ceutically acceptable carrier, wherein the pharma- ceutically acceptable carrier comprises a stabilizer. The stabilizer is Phe or a structural analog thereof. The stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid, and benzoic acid. A preferred range for the stabilizer of the present disclosure is about 0.1-20 moles of stabilizer per mole of prokaryotic PAL active site. A more preferred range for the stabilizer of the present disclosure is about 0.5-10 moles of stabilizer per mole of prokaryotic PAL active site. A most preferred range for the stabilizer of the present disclosure is about 1-10 moles of stabilizer per mole of prokaryotic PAL active site.

[0103] In some embodiments, the pharmaceutical composition comprises a prokaryotic PAL variant and a pharma- tically acceptable carrier, wherein the prokaryotic PAL variant has a higher phenylalanine conversion activity and / or a reduced immunogenicity compared to wild-type PAL, and is effective in reducing the Phe concentration in the blood, serum, or plasma of a subject from below the detection level to a range between about 20 μM and 60 μM, preferably below about 20 μM, and even more preferably below about 10 μM, and wherein the pharma-tically acceptable carrier comprises a stabilizer. In some embodiments, the stabilizer is Phe or a structural analog thereof. In some embodiments, the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid, and benzoic acid.

[0104] In some particular embodiments, the pharmaceutical composition comprises a prokaryotic PAL variant and a medicamentously acceptable carrier, wherein the prokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) variant, wherein the cysteine ​​residues at positions 503 and 565 of the AvPAL variant are replaced with serine residues, and the AvPAL variant further comprises a water-soluble polymer of polyethylene glycol, wherein the ratio of AvPAL variant to polyethylene glycol is about 1:3; and the AvPAL variant is effective to reduce the phenylalanine concentration in the blood, serum, or plasma of a subject from below the detection level to a range between about 20 μM to 60 μM, preferably to less than about 20 μM, and even more preferably to less than about 10 μM, and wherein the medicamentously acceptable carrier comprises a stabilizer. In some embodiments, the stabilizer is Phe or a structural analog thereof. In some embodiments, the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid, and benzoic acid.

[0105] In some more specific embodiments, the pharmaceutical compositions provided herein comprise an AvPAL variant comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and trans-cinnamic acid. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO:4. In some embodiments of the various pharmaceutical compositions described above, the pharmaceutical compositions further comprise sodium chloride, and tromethamine and tromethamine hydrochloride.

[0106] As used herein, the term "therapeutically effective amount" when contemplating a prokaryotic PAL variant composition means an amount effective to produce the intended beneficial effect on the health of a patient. In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant reduces blood, plasma or serum, preferably plasma L-phenylalanine levels to benefit the patient. The amount varies from individual to individual and depends on several factors, such as the patient's overall health, diet, and disease state. The amount of prokaryotic PAL used for therapy provides an acceptable reduction in blood, plasma or serum, preferably plasma L-phenylalanine levels and maintains this value at a beneficial level during PAL treatment (usually in the range of about less than 5% to between about 35%-100% of the normal range of blood, plasma or serum, preferably plasma L-phenylalanine, preferably in the range of about less than 5% to about 35%, and even more preferably in the range of about less than 5% to about 15%). In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant reduces tumor growth, tumor size, or tumor burden by more than about 10%, 30%, 50%, 70%, 90%, 95%, 98%, or 99% in treated patients compared to untreated patients. In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant maintains tumors in a static state in treated patients compared to untreated patients. In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant increases survival or disease-free intervals by at least about 10%, 20%, 50%, 100%, 2-fold, 5-fold, or 10-fold longer in treated patients compared to untreated patients. Therapeutically effective amounts of the prokaryotic PAL variant compositions of the present disclosure can be readily ascertained by one of skill in the art using publicly available materials and procedures.

[0107] The present disclosure provides for administering prokaryotic PAL variants less frequently than native PAL. The dosing frequency will vary depending on the condition being treated, but will generally be about once per week. It is understood that the dosing frequency actually used may vary somewhat from the frequencies disclosed herein due to the variability in the response of different individuals to prokaryotic PAL variants; the term "about" is intended to reflect such variability. It is envisioned that prokaryotic PAL variants are administered about twice per week, about once per week, about once every two weeks, about once per month, or longer than about once per month.

[0108] Thus, the present disclosure can be used to reduce blood, plasma, or serum L-phenylalanine levels. Numerous conditions in which depletion of blood, plasma, or serum L-phenylalanine levels would be beneficial can be treated with the prokaryotic PAL variant pharmaceutical compositions of the present disclosure.

[0109] Prokaryotic PAL pharmaceutical compositions prepared according to the present disclosure are preferably administered by parenteral injection, either intravenously, intraperitoneally, subcutaneously, intramuscularly, intraarterially, or intrathecally, however, it will be apparent to those skilled in the art that other delivery routes may also be effectively utilized with the pharmaceutical compositions of the present disclosure.

[0110] The methods described herein use prokaryotic PAL pharmaceutical compositions that include the above-mentioned molecules together with one or more pharma- ceutically acceptable excipients, vehicles, diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers, and, optionally, other therapeutic and / or prophylactic ingredients. Such excipients include liquids, such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins (i.e., sulfobutyl ether cyclodextrins), and the like. Excipients suitable for non-liquid formulations are also known to those of skill in the art.

[0111] Pharmaceutically acceptable salts can be used in the compositions of the present disclosure, including, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and organic acid salts such as acetate, propionate, malonate, benzoate, etc. A thorough discussion of pharma- ceutically acceptable excipients and salts is available in Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0112] Additionally, auxiliary substances such as wetting or emulsifying agents, biological buffer substances, surfactants, etc. may be present in such vehicles. The biological buffer can be virtually any solution that is pharmacologically acceptable and provides the formulation with a desired pH, i.e., a pH within the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, etc.

[0113] Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid, or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, creams, ointments, lotions, etc., and preferably in unit dosage forms suitable for single administration of precise dosage amounts. The compositions comprise a therapeutically effective amount of the prokaryotic PAL in combination with a pharma- ceutically acceptable carrier, and may, in addition, include other medicinal agents, adjuvants, diluents, buffers, etc.

[0114] In general, the prokaryotic PAL pharmaceutical compositions of the present disclosure will be administered in pharmaceutical formulations such as those suitable for oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous, and intravenous) administration, or in a form suitable for administration by inhalation or insufflation. The preferred mode of administration is intravenous, using a convenient daily dosage regimen that can be adjusted according to the severity of the affliction.

[0115] For solid compositions, conventional non-toxic solid carriers include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharma- ceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc., the prokaryotic PAL variant compositions described herein and optional pharmaceutical adjuvants in an excipient, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, thereby producing a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, tonicifying agents, e.g., sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, referenced above.

[0116] For oral administration, the composition generally takes the form of a tablet, capsule, or soft gel capsule, or may be an aqueous or non-aqueous liquid, suspension, or syrup. Tablets and capsules are suitable oral dosage forms. Tablets and capsules for oral use will generally contain one or more commonly used carriers, such as lactose and corn starch. Lubricants, such as magnesium stearate, are also generally added. When suspensions are used, the active agent may be combined with emulsifying or suspending agents. If desired, flavorings, colorings, and / or sweeteners may also be added. Other optional ingredients for incorporation into oral formulations herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.

[0117] Parenteral formulations can be prepared in any conventional form, either as a solution or suspension, a solid or lyophilized form suitable for reconstitution, solubilization, or suspension in liquid prior to injection, or as an emulsion. Preferably, sterile injectable suspensions are formulated by techniques known in the art using suitable carriers, dispersing or wetting agents, and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that can be employed include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils, fatty acid esters, or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a sustained release system or a sustained release system to maintain a constant level of dosage.

[0118] The prokaryotic PAL compositions of the disclosure described herein can be administered to a patient in a therapeutically effective dose. The toxicity and therapeutic efficacy of such prokaryotic PAL compositions can be determined, for example, by the LD 50 (the dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as by determining the dose that is therapeutically effective in 50% of a population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the ratio LD 50 / ED 50 Prokaryotic PAL compositions that exhibit large therapeutic indices are generally preferred.

[0119] (Identification and monitoring of patient populations) As described throughout this specification, various embodiments of the present disclosure may require determining whether a particular patient will respond to PAL therapy and determining the patient's phenylalanine concentration, both to initially identify the class of PKU patient being treated and to monitor the effectiveness of the ongoing treatment regimen during the course of the regimen. Exemplary such methods are described herein below.

[0120] (BH4 Loading Test) BH4 loading tests allow differentiation between patients with HPA caused by BH4 deficiency and those with HPA caused by PAH deficiency.

[0121] The simplest BH4 loading test is one in which exogenous BH4 is administered and the effect of the administration on lowering plasma Phe concentrations is determined. Initially, an intravenous loading of 2 mg / kg BH4 was proposed by Danks et al., Lancet 1:1236 (1976), and as higher purity BH4 has become available, it has become possible to perform studies using oral BH4 administration in amounts of approximately 2.5 mg / kg body weight. Finally, a standardized approach was proposed by Niederwieser et al., in which a single oral dose of 7.5 mg / kg BH4 is administered (Niederwieser et al., Eur. J. Pediatr. 138:441 (1982)). However, some institutions still use more than 20 mg BH4 / kg body weight.

[0122] For a simple BH4 loading test to give reliable results, the patient's blood Phe level must be higher than 400 μM. Therefore, in many cases, patients will discontinue the PKU diet for 2 days before performing the loading test. BH4 testing kits are available and distributed by Dr. Schircks' laboratory (Jona, Switzerland). The kit recommends a dosage of 20 mg BH4 / kg body weight administered approximately 30 minutes after ingestion of a regular meal.

[0123] (Determination of Phe concentration) Many methods exist for determining the presence of Phe in blood (Shaw et al., Analytical Methods in Phenylketonuria-Clinical Biochemistry, in Phenylketonuria and Some Other Inborn Errors of Amino Acid Metabolism, eds. Bickett et al., Stuttgart, Georg Thiem Verlag, 47-56 (1971)). Usually, phenylalanine and tyrosine concentrations are determined from the patient's serum using a fluorometric assay, which relies on the production of fluorescent substances when phenylalanine is heated with ninhydrin in the presence of leucylalanine (McCaman et al., J. Lab. Clin. Med. 59:885-890 (1962)).

[0124] The most widespread method for determining Phe concentrations is the Guthrie test, in which disks are punched from filter paper saturated with a patient-derived blood sample. The uniform disks are incubated in a tray of agar seeded with Bacillus subtilis and containing a specific inhibitor of B. subtilis growth. As phenylalanine migrates from the uniform disk onto the agar, Phe reverses the inhibition of bacterial growth, thereby producing a zone of bacterial growth that can be correlated to phenylalanine concentration by comparison with a similar assay performed with disks containing known amounts of Phe.

[0125] Other methods of quantifying Phe concentrations include HPLC, mass spectrometry, thin layer chromatography, etc. Such methods can be used to determine a patient's plasma Phe concentration prior to therapy and to monitor Phe concentrations during a treatment regimen to determine its effectiveness.

[0126] It is envisioned that patients will have their plasma Phe levels monitored at convenient intervals (e.g., daily, every other day, or weekly) throughout the time course of the treatment regimen. By such regular monitoring of plasma Phe levels, the clinician will be able to assess the effectiveness of treatment and adjust PAL and / or dietary protein requirements accordingly.

[0127] (Combination therapy) Certain methods of the invention include the combination of PAL and dietary protein restriction to produce therapeutic results in patients with various forms of HPA. To achieve appropriate therapeutic results in the combination therapy contemplated herein, the PAL composition will generally be administered to the subject and the dietary restriction will be implemented in a combined amount effective to produce the desired therapeutic result (i.e., a reduction in plasma Phe concentration and / or the ability to tolerate a greater amount of Phe / protein intake without a concomitant increase in plasma Phe concentration). This process may include administering the PAL composition and the dietary protein therapeutic composition simultaneously. This may be accomplished by administering a single composition or pharmacological protein formulation that contains all of the dietary protein requirement, with PAL also included within the protein formulation. Alternatively, the dietary protein (supplement or regular protein meal) is ingested at about the same time as the pharmacological formulation of PAL (tablet, injection, or drink). PAL may also be formulated as a protein bar or other food suitable for ingestion, such as brownies, pancakes, cakes, etc.

[0128] In other alternatives, PAL therapy may precede or follow dietary protein therapy with intervals ranging from minutes to hours. In embodiments where protein and PAL compositions are administered separately, one would generally ensure that no significant period of time had passed between the time of each delivery so that PAL could still exert beneficial effects on the patient. In such cases, it is contemplated that PAL would be administered within about 2-6 hours (before or after) dietary protein ingestion, with a time delay of only about 1 hour being most preferred. In certain embodiments, it is contemplated that PAL therapy would be a continuous therapy in which a daily dose of PAL is administered to the patient indefinitely. In other situations, for example, in pregnant women with only milder forms of PKU and HPA, PAL therapy would only be continued as long as the woman is pregnant and / or breastfeeding.

[0129] In addition to the therapy based only on the delivery of PAL and dietary protein restriction, the method of the present invention also contemplates combination therapy with a third composition that specifically targets one or more of the symptoms of HPA.For example, it is known that the tyrosine deficiency caused by HPA leads to a deficiency of neurotransmitters dopamine and serotonin.Therefore, in the context of the present invention, it is envisioned that the PAL and dietary protein-based method can be further combined with the administration of L-dopa, carbidopa, and 5-hydroxytryptophan neurotransmitters to correct the deficiency caused by the reduction in the amount of tyrosine in the diet.

[0130] Because administration of PAL (unlike administration of PAH) would not result in tyrosine, such treatment would still render tyrosine an essential amino acid for such patients, and therefore dietary supplementation with tyrosine would be desirable for patients receiving PAL in combination with BH4 therapy.

[0131] (Dietary Protein) In addition to administering the prokaryotic PAL composition to the subject, it is envisioned that the patient's dietary protein may also be restricted or altered.Those skilled in the art are aware of various commercially available protein formulations for use in the treatment of PKU.Such preparations include MAXIMAID, PHENEX 1, PHENEX 2 (Ross Laboratories, Liverpool, UK), LOFENALAC, PHENYL-FREE (Mead-Johnson), and the like.

[0132] Those skilled in the art may generally use the mentioned protein formulas that are free of Phe concentrations. Protein formulas are often supplemented with amino acids that are deficient in PKU patients. Such amino acids include, for example, L-tyrosine and L-glutamine.

[0133] Furthermore, it is known that L-carnitine and taurine, which are normally found in human breast milk and other foods of animal origin, should also be provided in addition to protein restriction. In one embodiment, L-carnitine can be provided as a 20mg / 100g protein supplement and taurine can be provided as a 40mg / 100g protein supplement to help provide the amounts of these factors normally found in human breast milk and foods of animal origin.

[0134] Additionally, one of skill in the art can refer to the 2000 National Academy of Sciences-National Research Council Dietary Reference Intakes for a further list of other ingredients, such as essential vitamins and minerals, that should be provided to the patient to ensure that other supplements are provided even when dietary protein is restricted.

[0135] With reference to the above discussion of total protein and desired plasma Phe concentrations, one skilled in the art would be able to determine the amount of dietary protein restriction required and adjust the patient's diet accordingly. Upon administration of prokaryotic PAL to the subject, determining whether the method of the present disclosure is effective would entail periodically determining the patient's plasma Phe concentration to ensure that the plasma Phe concentration remains in the range of below the level of detection to between about 20 μM and 60 μM, preferably below about 20 μM, and even more preferably below about 10 μM. Tests for determining such concentrations are described below. Preferably, concentrations below the level of detection to between about 20 μM and 60 μM are achieved, more preferably below about 20 μM, and even more preferably below about 10 μM.

[0136] In certain embodiments, the disclosure provides a method for treating a subject, comprising administering to a subject in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising a prokaryotic phenylalanine ammonia-lyase (PAL) variant and a pharma- ceutically acceptable carrier, wherein the PAL variant has higher phenylalanine converting activity and / or reduced immunogenicity compared to wild-type PAL and is effective to reduce a phenylalanine concentration in the subject's blood, serum, or plasma from below detection levels to a range of between about 20 μM and 60 μM, preferably to less than about 20 μM, and even more preferably to less than about 10 μM, and further comprising administering to the subject a protein-restricted (i.e., phenylalanine-free) diet.

[0137] To achieve a suitable therapeutic outcome in the combination therapy contemplated herein, the subject will generally be administered the prokaryotic PAL composition and dietary restriction in a combined amount effective to produce the desired therapeutic outcome (i.e., a reduction in plasma Phe concentration from below the level of detection to a range of optimally about 20 μM to 60 μM, preferably to less than about 20 μM, and even more preferably to less than about 10 μM, using standard detection methods well known in the art). This process may include administering the prokaryotic PAL composition and the dietary protein therapeutic composition simultaneously. This may be accomplished by administering a single composition or pharmacological protein formulation that includes all of the dietary protein requirement, which also includes the prokaryotic PAL within the protein formulation. Alternatively, the dietary protein (supplement or regular protein meal) is ingested at about the same time as the pharmacological formulation of prokaryotic PAL (tablet, injection, or drink). Prokaryotic PAL may also be formulated as a protein bar or other food suitable for ingestion, such as brownies, pancakes, cakes, etc.

[0138] Because administration of prokaryotic PAL (unlike administration of PAH) would not result in tyrosine, such treatment would still render tyrosine an essential amino acid for such patients, and therefore dietary supplementation with tyrosine would be desirable for patients receiving prokaryotic PAL alone or in combination with dietary protein therapy.

[0139] In other alternatives, the prokaryotic PAL treatment may precede or follow the dietary protein therapy with intervals ranging from minutes to hours. In embodiments where the protein and prokaryotic PAL compositions are administered separately, one will generally ensure that no significant period of time has elapsed between the time of each delivery so that the PAL can still exert a beneficial effect on the patient. In such cases, it is contemplated that the PAL will be administered within about 2-6 hours (before or after) of dietary protein ingestion, with a time delay of only about 1 hour being most preferred. In certain embodiments, it is contemplated that the PAL therapy will be a continuous therapy in which a daily dose of PAL is administered to the patient indefinitely.

[0140] (Production of Prokaryotic PAL) Another aspect of the invention is a method for producing prokaryotic PAL. In a preferred embodiment, recombinant PAL is overexpressed as an N-terminal octahistidyl tagged fusion protein in a vector with an inducible promoter such as one with IPTG (isopropyl-β-D-thiogalactopyranoside), preferably E. coli BL21 (DE3) / pLysS (Invitrogen). In another preferred embodiment, recombinant PAL is overexpressed in E. coli BL21 (DE3) / pLysS cells without an N-terminal tag. Seed cultures for bioreactors / fermentors are grown in shake flasks from glycerol stocks. Such seed cultures are then used to spike into bioreactors controlled in fed-batch mode. Glucose is added, pH is controlled with base (NH4OH), and agitation is at a maximum of 1200 rpm. Dissolved oxygen is kept above 20% by O2 feeding. Cells were cultured at an OD of 70-100. 600 The cells are grown at 30°C until an OD of 200 is reached (approximately 22-25 hours) and then induced with 0.4 mM IPTG. The temperature is lowered to 22-26°C until the activity changes to <0.1 IU / ml (approximately 40-48 hours and typically an OD of 200). 600The cells are grown to a viable yield of 1000 ng / ml. The cell culture medium is typically a defined medium, consisting of yeast extract proteins, peptone-tryptone, glucose, glycerol, casamino acids, trace salts and phosphate buffer salts. The recombinant PAL product is produced intracellularly and is not secreted. Bacteria are harvested by continuous centrifugation (Alfa-Laval, Carr, Ceba, or equivalent).

[0141] (Purification of prokaryotic PAL) A further aspect of the invention features a method for purifying bacterial PAL or a biologically active fragment, variant, or analog thereof. According to a first embodiment, the transformed cell mass is grown and disrupted, leaving behind the crude recombinant enzyme. Typically, extraneous material is separated from the crude bulk to prevent column contamination. Chromatographic purification is performed using one or more chromatography resins. The purified protein is then formulated in a buffer designed to provide stable activity over an extended period of time. In another preferred embodiment, the method for purifying bacterial PAL comprises: (a) lysis of bacteria containing recombinant PAL using a pressure homogenizer (but optionally by other physical means such as glass bead lysis); (b) heat treatment; (c) clarification of the lysate using a second successive centrifugation step and / or depth filtration (such as is performed using a Cuono Zeta Plus or Maximizer, Pall Filtron, or Millipore Millistak or Opticao filter); (d) passage through an activated charcoal filtration step (such as is performed using a Millipore Millistak 40AC); (e) passage through a final filtration step ( (f) passage through butyl hydrophobic interaction chromatography (as performed on a Toyopearl Butyl 650M from Tosoh Biosciences); (g) passage through a Q ion exchange column (as performed on a Macroprep High Q from BioRad); and (h) recovery of the final product by buffer exchange with tangential flow filtration (as performed with a Sartorious Hydrosart or PES 100 kDa membrane). Those skilled in the art will readily recognize that one or more of the chromatography steps may be omitted or substituted, or the order of the chromatography steps may be altered within the scope of the present invention. Finally, an appropriate sterilization step may be performed, if desired.

[0142] Having generally described the present invention, the present invention may be more easily understood through the following examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any manner. Some experimental error and deviations should, of course, be taken into account when trying to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.).

[0143] Therapeutic Uses and Administration Various forms of hyperphenylalaninemia (HPA)

[0144] Provided herein are methods for treating various HPA patient populations, including the use of the pharmaceutical compositions provided herein, either alone or in combination with other treatment regimens, to manage HPA and / or PKU.In particular, it is envisioned that the pharmaceutical compositions provided herein can be used to treat patient populations with phenylalanine concentrations so low that dietary intervention is not normally used (i.e., patients with mild HPA), patients with moderate PKU, patients with classical or severe PKU, and any subpopulations thereof.

[0145] A particular embodiment is directed to treating classical severe PKU by administering to a subject a protein-restricted diet in combination with a composition comprising a prokaryotic PAL variant or a bioactive variant, mutant, or fragment thereof, wherein the combined administration of the protein-restricted diet and the prokaryotic PAL variant is effective to reduce the phenylalanine concentration in the plasma of the subject, compared to the concentration in the absence of the combined administration.In a particular embodiment, the therapy is contemplated for a patient with a Phe level of more than 420 μM.In another particular embodiment, the therapy is contemplated for a patient with a Phe level of more than 500 μM.In yet another particular embodiment, the therapy is contemplated for a patient with a Phe level of more than 550 μM.In yet another particular embodiment, the therapy is contemplated for a patient with a Phe level of more than 600 μM.In a particular embodiment, the therapy is contemplated for a patient with a Phe level of more than 650 μM.

[0146] Other embodiments involve administering a pharmaceutical composition comprising a prokaryotic PAL variant provided herein to any individual with HPA characterized by a plasma Phe concentration greater than 180 μM prior to administration of the prokaryotic PAL variant, in an amount effective to cause a reduction in such plasma Phe concentration in the patient.

[0147] Characteristics of severe classical PKU and its treatment

[0148] Severe PKU manifests itself in plasma Phe concentrations of over 1200 μM and may be seen as high as 4800 μM. Patients with this disorder must be treated with a Phe-free diet to reduce plasma Phe concentrations to clinically acceptable levels (usually less than 600 μM or less than 300 μM). These patients can only tolerate up to 250-350 mg of dietary Phe per day (Spaapen et al., Mol. Genet Metab. 78:93-99 (2003)). Thus, these patients are placed on a Phe-restricted prescription diet starting 7-10 days after birth, and this dietary restriction is imposed for the remainder of the patient's life. Any relief from the strict dietary restrictions imposed on these individuals would be beneficial.

[0149] The tests used for the diagnosis of individuals with classical Phe are described in more detail herein. These tests reveal that patients with classical severe PKU require a low phenylalanine diet (Lucke et al., Pediatr. Neurol. 28:228-230 (2003)). It is therefore envisioned that certain methods provided herein will entail determining that a patient suffers from classical PKU by monitoring the individual's plasma Phe concentration. The patient can then be treated by administering a pharmaceutical composition comprising a prokaryotic PAL variant provided herein alone or a combination regimen of a low protein diet and a PAL variant, so as to result in at least a 25% reduction in the patient's plasma Phe concentration. In some embodiments, the method will result in a 30% reduction in plasma Phe concentration. In another embodiment, the method will result in a 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in an individual's plasma Phe concentration (e.g., if a patient with severe classical PKU has a Phe concentration of 4800 μM, a 90% reduction in Phe concentration will result in a plasma Phe concentration of 480 μM, a concentration low enough that little dietary restriction is required). Of course, it should be understood that the therapeutic methods provided herein, whether for treating severe classical PKU or any other HPA described herein, should attempt to reduce the patient's plasma Phe concentration to a level as close as possible to the range of about 120 μM to about 360 μM ± 15 μM or the optimal range of about 120 μM to about 240 μM.

[0150] In some embodiments, the plasma Phe concentration of a PKU patient on classical treatment is reduced from any amount of unrestricted plasma Phe concentration above 1000 μM to any plasma Phe level below 600 μM. Of course, even if combined treatment with a prokaryotic PAL variant and a protein-restricted diet results in a smaller reduction in plasma Phe concentration, for example to a level of 800 μM to about 1200 μM, this would be considered a clinically useful outcome of the therapy because patients with plasma Phe concentrations in this range would be able to manage their disease simply by restricting the amount of protein in their diet as opposed to taking a Phe-restricted regimen, thereby resulting in a significant improvement in the individual's quality of life as well as improved compliance of the patient with the dietary restrictions.

[0151] Any increase in the amount of dietary Phe levels that a patient can tolerate as a result of the treatment would be considered a therapeutically beneficial outcome. For example, it is envisioned that as a result of administering a prokaryotic PAL variant therapy, a patient would be able to increase their dietary Phe intake from 250-350 mg / day to 350-400 mg / day (i.e., the patient's Phe resistance phenotype would be altered from that of a classical PKU patient to that of a moderate PKU patient). The therapeutic intervention taught herein would desirably enable a patient to increase their dietary Phe intake from 250-350 mg / day to 400-600 mg / day (i.e., the patient's Phe resistance phenotype would be altered from that of a classical PKU patient to that of a mild PKU patient), or in some cases, enable the patient to have an intake (i.e., normal dietary intake) of more than 600 mg Phe / day.

[0152] Characteristics of patients with BH4-unresponsive PKU and methods of treating same

[0153] A second group of patients that can be treated with the pharmaceutical compositions and methods provided herein are individuals who have been determined to have elevated plasma Phe concentrations, i.e., any concentration above 200 μM, but have been diagnosed as non-responsive to BH4 therapy (as determined by BH4 loading testing, described below). Such patients can include individuals with mild PKU (i.e., plasma Phe concentrations up to 600 μM), individuals with moderate PKU (i.e., plasma Phe concentrations between 600 μM and about 1200 μM), and patients with classical severe PKU (i.e., plasma Phe concentrations above 1200 μM).

[0154] In some embodiments, a patient who is non-responsive to BH4 therapy is given PAL variant in combination with a reduction in the amount of protein in the patient's diet to reduce the patient's plasma Phe concentration.The administration of prokaryotic PAL variant can cause a greater reduction in the patient's plasma Phe concentration compared to the reduction caused by the same dietary protocol carried out in the absence of prokaryotic PAL variant therapy.The dietary restriction can be a diet that restricts Phe intake by providing a synthetic medical protein formula with a reduced amount of Phe, or the dietary restriction can simply require the patient to restrict its total protein intake, but still allow the patient to eat normal food in a limited amount.

[0155] The therapeutic outcomes described for classical PKU patients are incorporated herein by reference. For example, for patients with moderate PKU (i.e., patients with unrestricted plasma Phe concentrations between 600 μM and 1200 μM), therapeutic outcomes may include at least a 25% reduction in the patient's plasma Phe concentration. In some embodiments, the method will result in a 30% reduction in plasma Phe concentration. In other embodiments, the method will result in a 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in the individual's plasma Phe concentration (e.g., if a patient with moderate classical PKU has a Phe concentration of 1000 μM, a 90% reduction in Phe concentration will result in a plasma Phe concentration of 100 μM, a concentration low enough that little or no dietary restriction is required).

[0156] In some embodiments, the plasma Phe concentration of moderate PKU patients undergoing treatment is reduced from an unrestricted plasma Phe concentration of any amount between 600 μM and 1200 μM to any plasma Phe level that is less than 300 μM. In one embodiment, treatment with prokaryotic PAL variants (either alone or in combination with dietary restriction) results in a reduction in plasma Phe concentration, for example, to a level between 200 μM and about 400 μM. This would be considered a clinically useful outcome of therapy because patients with plasma Phe concentrations in this range can manage their disease simply by restricting the amount of protein in their diet, as opposed to taking a Phe-restricted formula. In fact, many studies have taught that such patients can even eat a normal diet.

[0157] An increase in the amount of dietary Phe levels that a patient can tolerate as a result of the treatment would be considered a therapeutically effective outcome. For example, it is envisioned that as a result of administering prokaryotic PAL variant therapy (either alone or in combination with other therapeutic interventions), a patient would be able to increase their dietary Phe intake from 350-400 mg / day to 400-600 mg / day (i.e., the patient's Phe resistance phenotype would be altered from that of a moderate PKU patient to that of a mild PKU patient). Of course, it would be desirable for the therapeutic intervention taught herein to enable a patient to increase their dietary Phe intake from 350-400 mg / day to have an intake of more than 600 mg Phe / day (i.e., normal dietary intake).

[0158] Patients who exhibit only mild PKU, i.e., have a dietary tolerance of 400-600 mg Phe intake / day, can be treated using the compositions and methods provided herein and can benefit from prokaryotic PAL variant-based therapy. This is because it is desirable to result in a normalized plasma Phe concentration as close as possible to 360 μM±15 μM. For such patients, a favorable therapeutic outcome would include at least a 25% reduction in the patient's plasma Phe concentration. In one embodiment, the method would result in a 30% reduction in plasma Phe concentration. In another embodiment, the method would result in a 40%>, 50%>, 60%>, or greater reduction in the individual's plasma Phe concentration (e.g., if a patient with mild PKU has a Phe concentration of 600 μM, a 60% reduction in Phe concentration would result in a plasma Phe concentration of 360 μM, i.e., an acceptable normal plasma Phe concentration).

[0159] In some embodiments, the plasma Phe concentration of a mild PKU patient undergoing treatment is reduced from any amount of unrestricted plasma Phe concentration between 400 μM and 600 μM to any plasma Phe level that is less than 100 μM. Of course, even if treatment with a prokaryotic PAL variant (either alone or in combination with dietary restriction) results in a smaller reduction in plasma Phe concentration, for example, to a level of 200 μM to about 400 μM, this would still be considered a clinically useful outcome of the therapy.

[0160] Any increase in the amount of dietary Phe levels that a patient can tolerate as a result of the treatment would be considered a therapeutically beneficial outcome. For example, it is envisioned that as a result of administering prokaryotic PAL variant therapy (either alone or in combination with other therapeutic interventions), a patient would be able to increase their dietary Phe intake from 400-600 mg / day (i.e., the patient's Phe resistance phenotype would be changed from that of a mild PKU patient to that of a mild HPA patient), allowing the patient to have an intake (i.e., normal dietary intake) of more than 600 mg Phe / day.

[0145] Furthermore, even patients who only exhibit non-PKU HPA symptoms, i.e., have elevated plasma Phe concentrations up to 600 μM, but are otherwise permitted to consume a normal protein diet, would still benefit from prokaryotic PAL variant therapy. This is because elevated Phe concentrations have been shown to have a significant effect on the IQ of such individuals.

[0161] For the sake of brevity, certain abbreviations are used herein. One example is the one-letter abbreviations that represent amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: [Table 1]

[0162] The present invention is generally disclosed herein using categorical language to describe a number of embodiments. The present invention also expressly includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays, or analyses, is excluded in whole or in part. Thus, even if the present invention is not generally set forth herein in terms of what the present invention does not include, aspects not explicitly included in the present invention are nevertheless disclosed herein.

[0163] Certain embodiments of the present invention, including the best mode known to the inventors for carrying out the invention, are described herein. Upon reading the above description, variations of the disclosed embodiments will become apparent to those working in the art, and it is expected that such variations will be adopted by those skilled in the art as appropriate. It is therefore intended that the present invention be practiced otherwise than as specifically described herein, and that the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention, unless otherwise indicated herein or clearly contradicted by context.

[0164] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety, unless and to the extent that it is inconsistent with this disclosure, and to the same extent as if each individual publication, patent application, patent, or reference was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by reason of prior invention. Further, the dates of publications listed may be different from the actual publication dates, which may need to be independently confirmed.

[0165] (8. Implementation) (Embodiment 1) 1. A method for reducing blood phenylalanine levels in a subject, comprising: administering to the subject a weekly dose of a formulation comprising an AvPAL variant; The subject is about 12 to about 18 years old, and the weekly dose is administered for more than about 50 weeks; The method, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0166] (Embodiment 2) 2. The method of embodiment 1, wherein said weekly dose is administered for more than about 60 weeks, more than about 70 weeks, more than about 80 weeks, more than about 90 weeks, more than about 100 weeks, more than about 110 weeks, more than about 120 weeks, more than about 130 weeks, more than about 140 weeks, more than about 150 weeks, more than about 160 weeks, more than about 170 weeks, more than about 180 weeks, more than about 190 weeks, more than about 200 weeks, more than about 210 weeks, more than about 220 weeks, more than about 230 weeks, more than about 240 weeks, or more than about 250 weeks.

[0167] (Embodiment 3) The method of embodiment 1 or 2, wherein the dosage ranges from about 0.1 mg per week to about 1 mg per week.

[0168] (Embodiment 4) The method of embodiment 1 or 2, wherein the dosage ranges from about 1 mg per week to about 2 mg per week.

[0169] (Embodiment 5) The method of embodiment 1 or 2, wherein the dosage ranges from about 2 mg per week to about 10 mg per week.

[0170] (Embodiment 6) The method of embodiment 1 or 2, wherein the dosage ranges from about 10 mg per week to about 20 mg per week.

[0171] (Embodiment 7) The method of embodiment 1 or 2, wherein the dosage ranges from about 20 mg per week to about 40 mg per week.

[0172] (Embodiment 8) The method of embodiment 1 or 2, wherein the dosage ranges from about 40 mg per week to about 70 mg per week.

[0173] (Embodiment 9) The method of embodiment 1 or 2, wherein the dosage ranges from about 70 mg per week to about 140 mg per week.

[0174] (Embodiment 10) The method of embodiment 1 or 2, wherein the dosage ranges from about 140 mg per week to about 280 mg per week.

[0175] (Embodiment 11) The method of embodiment 1 or 2, wherein the dosage ranges from about 280 mg per week to about 420 mg per week.

[0176] (Embodiment 12) The method of embodiment 1 or 2, wherein the dosage ranges from about 420 mg per week to about 840 mg per week.

[0177] (Embodiment 13) The method of any one of embodiments 1-12, wherein the AvPAL variant is administered once a week.

[0178] (Embodiment 14) The method of any one of embodiments 1-12, wherein the AvPAL variant is administered twice weekly.

[0179] (Embodiment 15) The method of any one of embodiments 1-12, wherein the AvPAL variant is administered four times per week.

[0180] (Embodiment 16) The method of any one of embodiments 1-12, wherein the AvPAL variant is administered 7 times per week.

[0181] (Embodiment 17) The method of any one of embodiments 1-12, wherein the AvPAL variant is administered 14 times per week.

[0182] (Embodiment 18) The method of any one of embodiments 1-12, wherein the AvPAL variant is administered daily.

[0183] (Embodiment 19) (1) administering to the subject the AvPAL variant at an induction dosage ranging from about 0.1 mg per week to about 10 mg per week, followed by (2) administering to the subject the AvPAL variant in a titrated dosage ranging from about 1 mg per week to about 200 mg per week; followed by (3) administering to the subject the AvPAL variant at a maintenance dosage ranging from about 20 mg per week to about 840 mg per week. 2. The method of embodiment 1, comprising:

[0184] (Embodiment 20) The method of embodiment 19, wherein the induction dosage is about 2.5 mg per week.

[0185] (Embodiment 21) The method of embodiment 19 or 20, wherein the titration dosage ranges from about 5 mg per week to about 70 mg per week.

[0186] (Embodiment 22) The method of any one of embodiments 19-21, wherein the maintenance dosage is in the range of about 140 mg per week to about 420 mg per week.

[0187] (Embodiment 23) The induction dosage is administered for a period of about 2 weeks to about 6 weeks, the titration dose is administered for a period of about 3 weeks to about 8 weeks; and The maintenance dose is administered for a period of about 50 weeks to about 80 weeks. A method according to any one of embodiments 19 to 22.

[0188] (Embodiment 24) the induction dose is administered for a period of about 4 weeks; the titration dose is administered for a period of about 5 weeks; and The maintenance dose is administered for a period of about 56 to 64 weeks. The method of embodiment 23.

[0189] (Embodiment 25) The method of embodiment 23, wherein the maintenance dosage amounts comprise a first maintenance dosage amount of about 70 mg per week to about 280 mg per week, a second maintenance dosage amount of about 140 mg per week to about 560 mg per week, and a third maintenance dosage amount of about 210 mg per week to about 840 mg per week.

[0190] (Embodiment 26) the first maintenance dose is administered for a period of about 16 weeks to about 24 weeks; the second maintenance dose is administered for a period of about 16 weeks; and wherein the third maintenance dose is administered for a period of about 24 weeks. The method of embodiment 25.

[0191] (Embodiment 27) The method of any one of embodiments 19 to 26, further comprising administering to the subject, after administration of the maintenance dosage, an extended dosage of the AvPAL variant in the range of about 20 mg per week to about 840 mg per week.

[0192] (Embodiment 28) The method of embodiment 27, wherein the extended dosage is administered for a period of about 40 weeks to about 120 weeks.

[0193] (Embodiment 29) the induction dose is administered for a period of about 4 weeks; said titration dose being administered for a period of about 5 weeks; said maintenance dose is administered for a period of about 64 weeks; and The extended dose is administered for a period of about 80 weeks. The method of embodiment 28.

[0194] (Embodiment 30) The method of any one of embodiments 19-29, further comprising assessing the blood phenylalanine concentration prior to administering the induction dose.

[0195] (Embodiment 31) The method of any one of embodiments 19 to 30, further comprising evaluating the blood phenylalanine concentration after administration of one or more induction doses, titration doses, maintenance doses, and / or extension doses.

[0196] (Embodiment 32) 32. The method of embodiment 31, further comprising adjusting the dosage based on the blood phenylalanine concentration.

[0197] (Embodiment 33) The method of embodiment 32, wherein the dosage is adjusted to achieve a blood phenylalanine concentration of less than about 600 μM.

[0198] (Embodiment 34) The method of embodiment 32, wherein the dosage is adjusted to achieve a blood phenylalanine concentration of less than about 360 μM.

[0199] (Embodiment 35) The method of embodiment 33, wherein the maintenance dosage is increased if the blood phenylalanine concentration is greater than about 360 μM.

[0200] (Embodiment 36) The method of any one of embodiments 1 to 35, wherein the subject is suffering from phenylketonuria (PKU).

[0201] (Embodiment 37) The method of any one of embodiments 1 to 36, wherein the subject is about 12 to about 15 years old.

[0202] (Embodiment 38) The method of any one of embodiments 1 to 36, wherein the subject is about 16 to about 17 years old.

[0203] (Embodiment 39) The method of any one of embodiments 1 to 38, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:2.

[0204] (Embodiment 40) The method of any one of embodiments 1 to 38, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:3.

[0205] (Embodiment 41) The method of any one of embodiments 1 to 38, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:4.

[0206] (Embodiment 42) The method of any one of embodiments 1-41, wherein the AvPAL variant is pegylated.

[0207] (Embodiment 43) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of at least 1.6 polyethylene glycol per lysine residue of the AvPAL variant.

[0208] (Embodiment 44) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of at least 2.4 polyethylene glycol per lysine residue of AvPAL variant.

[0209] (Embodiment 45) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of three polyethylene glycol per lysine residue of the AvPAL variant.

[0210] (Embodiment 46) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 5 polyethylene glycol per lysine residue of the AvPAL variant.

[0211] (Embodiment 47) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of six polyethylene glycol per lysine residue of the AvPAL variant.

[0212] (Embodiment 48) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 7 polyethylene glycol per lysine residue of the AvPAL variant.

[0213] (Embodiment 49) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 8 polyethylene glycol per lysine residue of the AvPAL variant.

[0214] (Embodiment 50) The method of embodiment 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 9 polyethylene glycol per lysine residue of the AvPAL variant.

[0215] (Embodiment 51) The method of any one of embodiments 1-50, wherein the AvPAL variant is administered in a formulation comprising a pharma- ceutically acceptable carrier that comprises a stabilizer.

[0216] (Embodiment 52) 52. The method of embodiment 51, wherein the stabilizer is L-phenylalanine or a structural analog thereof.

[0217] (Embodiment 53) The method of embodiment 52, wherein the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid, and benzoic acid.

[0218] (Embodiment 54) The method of embodiment 53, wherein the stabilizer is trans-cinnamic acid.

[0219] (Embodiment 55) The method of embodiment 54, wherein the formulation further comprises sodium chloride, and tromethamine and tromethamine hydrochloride.

[0220] Several embodiments of the present invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the experimental section are intended to be illustrative, but not limiting, of the scope of the invention as defined in the claims. EXAMPLES

[0221] 9. Working Examples 9.1 Example 1: Effects of AvPAL variants on subjects aged 16-17 years A study was conducted to investigate the effect of a pegylated form of an AvPAL polypeptide variant (e.g., one having serine substitutions of the cysteine ​​residues at positions 503 and 565 (SEQ ID NO: 4)) in adolescent / young adult PKU patients aged 16-17 years.

[0222] A method for preparing the pegylated AvPAL double cysteine ​​mutant AvPAL_C565SC503S is described in commonly owned US7534595B2, the entirety of which is incorporated herein by reference. The pegylated AvPAL double cysteine ​​mutant AvPAL_C565SC503S was prepared as described in Example 10 of US7534595B2.

[0223] The study designs for PRISM-1 (165-301, NCT01819727) and PRISM-2 (165-302, NCT01889862) have been reported previously (Thomas JA et al., Mol Genet Metab. 2018;124(1):27-38). Safety, efficacy, and immunogenicity of PEG-valiase were evaluated in 11 subjects who were 16 or 17 years of age at the time of consent.

[0224] (Exposure and nature of subject) The exposure and disposition to PEGylalase for the 11 adolescent / young adult subjects in PRISM-2 was similar to that of the adult (>= 18 years) population. Baseline demographics and characteristics are shown in Table 1. Table 1. Baseline demographics and characteristics of subjects participating in PRISM-1 by age. [Table 2]

[0225] The mean (SD) duration of exposure for adolescent / young adult subjects was 885.5 (645.06) days, and the mean (SD) daily dose was 36.9 (12.70) mg / day. Most adolescent / young adult subjects received a mean dose of ≥ 40 mg / day to < 60 mg / day (45.5% [n = 5]) or ≥ 20 mg / day to < 40 mg / day (36.4% [n = 4]), 18.2% (n = 2) received a mean dose of < 20 mg / day, and none received a mean dose of ≥ 60 mg / day. Four of 11 adolescent / young adult subjects (36%) discontinued study drug: two (18%) due to AEs, one (9%) discontinued study drug by investigator decision (due to subject noncompliance), and one (9%) was lost to follow-up.

[0226] (Efficacy Results) Similar to adults, adolescents / young adults receiving chronic PEG-valiase therapy in PRISM-2 Part 4 were able to achieve clinically meaningful plasma Phe thresholds (<600 μmol / L [European guideline target for patients aged >12 years], <360 μmol / L [American College of Medical Genetics and Genomics (ACMG) target for all patients], <120 μmol / L [upper limit of normal]) (Figures ​(Figure1A-C)) and demonstrated substantial and sustained reductions in mean plasma Phe over time, with mean (SD) 595.8 (539.07) and 500.0 (625.01) μmol / L by week 49 and week 169, respectively, which represented reductions from baseline Phe levels of 36.8% and 47.1%, respectively.

[0227] (Safety / immunogenicity results) Safety and immunogenicity were evaluated by determining the rate of adverse events by age group for subjects enrolled in PRISM-2. The results are shown in Table 2. Adverse events (AEs) occurred at similar rates in both age groups (Table 2). None of the acute systemic hypersensitivity reactions were drug-specific Immunoglobulin E-related, and all events resolved without sequelae. Neither of the two adolescent / young adult subjects who experienced a serious adverse event (SAE) discontinued the study drug or study due to the event. Immunogenicity and pharmacokinetic / pharmacodynamic (PK / PD) profiles were consistent between the two age groups. PAL IgG antibodies were identified in 100% of subjects, neutralizing antibodies (NAbs) were identified in the majority of subjects, and mean detectable levels remained stable or decreased over time in both age groups. Table 2. Summary of adverse event rates by age group in subjects enrolled in PRISM2 [Table 3]

[0228] Results indicate that adolescent / young adult subjects aged 16-17 years achieved substantial and sustained plasma Phe reductions with PEGvaliase dosages up to 60 mg / day, with a manageable safety profile for the majority of subjects treated long-term. Efficacy, safety, and immunogenicity results in adolescent / young adults were consistent with those seen in adults, demonstrating a positive benefit:risk profile and supporting the inclusion of adolescent / young adult PKU patients aged 16-17 years on treatment with PEGvaliase. As adherence to dietary management begins to deteriorate during adolescence, pharmacotherapy should be considered to achieve optimal plasma Phe control in this patient population.

[0229] 9.2 Example 2: Clinical Evaluation of Prokaryotic PAL Compositions for the Treatment of Adolescents Ages 12-17 Phenylketonuria (PKU) is a rare autosomal recessive genetic disorder associated with absent or insufficient phenylalanine hydroxylase activity. The resulting elevation of blood phenylalanine (Phe) can cause cognitive neurochemical and psychiatric symptoms. Lifelong dietary management to maintain Phe below the recommended threshold is therefore important. Difficulties in maintaining Phe are commonly reported in adolescents as they leave childhood and have less parental supervision over dietary management. PEG-valiase is a pegylated PAL enzyme replacement therapy approved to lower Phe in adults with PKU (Phe>600 μmol / L). The following examples provide guidance on parameters used for clinical evaluation of compositions comprising prokaryotic PAL or a bioactive fragment, mutant, variant, or analog thereof in the therapeutic methods of the present disclosure. A Phase 3, open-label, randomized controlled trial is designed to evaluate the safety and efficacy of PEG-valiase in adolescents (NCT05270837). As discussed throughout this specification, prokaryotic PAL compositions are used to treat adolescent subjects ages 12-17. A clinical trial will be conducted that provides an evaluation of subcutaneous doses of prokaryotic PAL for safety, pharmacokinetics, and early response to both surrogate and defined clinical endpoints.

[0230] (method) This study is conducted to evaluate the safety and efficacy of self-administered subcutaneous injections of PEG-variase in adolescent subjects (ages 12-17) with phenylketonuria (PKU). Adolescent subjects with PKU aged 12-17 years will be divided into two age cohorts (Cohort A: 16-17 years and Cohort B: 12-15 years). Cohorts A and B will be enrolled and administered in parallel (see Figure 2A-C).

[0231] Cohort A will consist of approximately 25 U.S. subjects ages 16-17 years who will be evaluated using an open-label, single-arm study design. The primary efficacy outcome measure will be change from baseline in blood Phe at week 73, the end of the primary treatment phase (part 1). After week 73, cohort A subjects will continue to receive open-label PEGValiase for up to an additional 80 weeks in the extension phase (part 2).

[0232] Cohort B will consist of 27 US and European subjects aged 12-15 years (inclusive) who will be evaluated using an open-label, two-arm randomized controlled design with dietary management of PKU alone as the control. Subjects will be randomized in a 2:1 ratio to active and control arms, including 18 subjects who will receive PEGValiase and 9 subjects who will manage their PKU with dietary management alone, respectively. The primary efficacy outcome measure will be the change from baseline in plasma Phe at the end of part 1, week 73. After week 73, the 18 subjects in the active treatment arm will continue to receive open-label PEGValiase for up to an additional 80 weeks in the extension phase (part 2). Nine subjects in the diet-only control arm will begin treatment with pegvaliase at week 74 and from week 74 to week 146 will follow the same dosing and evaluation schedule that active subjects in both Cohort A and Cohort B followed from week 1 to week 73.

[0233] The two-part, randomized, targeted Phage 3 clinical trial will evaluate the risks and benefits of PEGValiase compared to dietary restriction alone in adolescents with needs that are not substantially met by current treatment options. The study will enroll approximately 54 adolescents with Phe >600 μmol / L (ages 12-17 years (US) inclusive; ages 12-15 years (EU) inclusive). In part 1, participants will be randomized 2:1 to PEGValiase (n=36) or dietary management (n=18) and followed for 72 weeks. After a 4-week lead-in period followed by a titration period, the maintenance dose of PEGValiase will be individualized up to a maximum of 60 mg / day depending on patient response. The primary efficacy endpoint will be change from baseline in blood Phe after 72 weeks of study. All participants in Part 1 will continue into Part 2 (starting at Week 73) and will receive pegvaliase treatment through Week 153. In addition to safety and Phe endpoints, cognitive neuroscience changes will be assessed.

[0234] Design rationale for separate cohorts The rationale for using two distinct designs to study older (16-17 years) and younger (12-15 years) adolescents in a single, all-inclusive study was based on an evaluation of how to rapidly meet the unmet medical need in all adolescent PKU patients within a single study.

[0235] Based on extensive clinical experience evaluating both sapropterin dihydrochloride and PEG-valiase in PKU patients, to assess blood Phe efficacy in the PKU population, dietary protein intake is maintained uniformly throughout the study in an open-label design with each subject serving as his or her own control. Within-subject comparisons to baseline blood Phe are warranted as evidence of efficacy since they take into account substantial inter-subject variability due to each subject's individual dietary Phe tolerance (i.e., the amount of dietary Phe an individual can consume daily while maintaining blood Phe concentrations within a defined target range). Phe tolerance may be determined by physiological attributes such as residual PAH activity, which is genetically determined and stable, in addition to the subject's catabolic / anabolic status.

[0236] For cohort A, consisting of older adolescent PKU patients (ages 16-17 years) enrolled in a US center, the open-label, single-arm design enhances the safety evaluation of PEGValiase by allowing a larger safety database for a given study size, since all subjects receive active treatment from the start of the study. This is achieved without the loss of safety insight that often occurs in randomized controlled trial (RCT) designs. Type III immune complex-mediated hypersensitivity events, which largely define the safety profile of PEGValiase, are not commonly observed in adult or adolescent PKU patient populations not receiving PEGValiase. As a result, comparison of safety data between PEGValiase treatment and control groups (whether dietary restriction alone or blinded placebo) is unlikely to reveal safety signals that would not be evident in an open-label study. Because the immune system matures prior to adolescence (Olin et al., Cell. 174(5):1277-1292.e14 (2018); Georgountzou et al., Front Immunol. 8:957 (2017)), the immune response to PEGvaliase among adolescent subjects is expected to be similar to that seen in study subjects aged 16-57 years in the PEGvaliase clinical development program. Importantly, 12 subjects from the 16-17 year old age group have been studied in previous PEGvaliase clinical trials, as described in Example 1, and data from these subjects indicates that this adolescent population is similar to adults with respect to both safety and efficacy.

[0237] In cohort B, consisting of young adolescent PKU patients (aged 12-15 years) enrolled in centers in both the EU and the US, a randomized controlled trial (RCT) design using dietary management alone of PKU as the control ensures robust data in a young, previously untested adolescent population and minimizes potential bias. Daily placebo injections would overburden adolescent PKU patients, so the dietary control arm is the best control for this age group. The dietary control addresses the possible effect of age on response due to possible adult behavioral differences related to compliance with the diet. Subjects randomized to the dietary control will continue their current prescribed diet.

[0238] The adolescent subjects of cohorts A and B are effectively participating in two separate cohorts under a single all-inclusive protocol. Given that the study procedures, dosing and evaluation schedules, safety monitoring, and other operational aspects of the study for cohorts A and B are essentially the same, this is done in an attempt to address unmet needs in adolescent PKU patients as efficiently as possible. Combining the cohorts into one study ensures consistency in study procedures, locations, and key personnel, which ensures more consistent evaluation across the two cohorts.

[0239] (Rationale for Dosing Regimen) PEGValiase is a routine treatment for patients with PKU. Previous clinical trials have suggested that the primary clearance mechanism of PEGValiase from plasma is via the formation of circulating immune complexes leading to the removal of the drug by complement activation and phagocytosis, indicating that plasma exposure of PEGValiase is determined by the immune response. Given that the immune system matures before adolescence (Olin et al., Cell. 174(5):1277-1292.e14 (2018); Georgountzou et al., Front Immunol. 8:957 (2017)), the immune response to PEGValiase among adolescent subjects is expected to be similar to that observed in test subjects aged 16-57 years in our clinical development program.

[0240] After dosing of the initial dose in the clinic (first 8 weeks), PEGvaliase will be self-administered by the subject under the observation of a parent or guardian (trained adult observer). Self-administration at home will make daily treatment less burdensome for the patient, thereby improving treatment compliance. In addition to training in self-administration prior to the first clinic dose, subjects and trained adult observers will receive extensive training on how to monitor for and respond to potential adverse events (AEs) that may be associated with treatment with the investigational drug.

[0241] Pegvaliase is administered using an induction / titration / maintenance (I / T / M) dosing regimen according to Table 3 (modified from the regimen used in Phase 3 trials conducted in the United States and the United States Prescribing Information (USPI)). During clinical development of Pegvaliase, this dosing regimen was found to help reduce the incidence and severity of hypersensitivity reactions while substantially reducing blood Phe concentrations.

[0242] The PEGvaliase dose levels in this study have been evaluated in six multiple-dose Phase 2 and Phase 3 studies. The dose level for induction was 2.5 mg once weekly, chosen because this dose, administered at a weekly dosing frequency for the first 4 weeks of treatment (i.e., induction), has been associated with a reduced incidence and severity of hypersensitivity reactions compared to a higher starting dose (0.4 mg / kg) at a dosing frequency of 5 days per week.

[0243] The 73-week duration for Part 1 was chosen because it was the amount of time it would be expected to take subjects to reach a 60 mg / day dose level following the protocol-specified dosing schedule to achieve optimal blood Phe levels, if needed.

[0244] (Objectives and Evaluation Items (Cohorts A and B)) The primary objective is to evaluate the safety and efficacy of PEG-valiase in adolescent subjects with PKU.

[0245] For Cohort A, the primary efficacy assessment is the change in blood Phe concentration from treatment-naïve baseline to Week 73. For Cohort A (ages 16-17), subjects will serve as their own controls. Subjects will be assessed for blood Phe concentration during Screening / Run-in (2 measurements 2-4 weeks apart), pre-dose on Day 1, every 4 weeks until the end of the Primary Treatment Phase (Part 1), and every 8 weeks in the Extension Phase (Part 2). The primary efficacy assessment is the change in blood Phe concentration from treatment-naïve baseline at Week 73.

[0246] For Cohort B (ages 12-15 years), change from baseline in blood Phe will be compared between subjects in the active (PEGylation) arm and the control (dietary restriction alone) arm. Subjects will be assessed for blood Phe concentrations during screening / observation (2 measurements 2-4 weeks apart), pre-dose on Day 1, every 4 weeks until the end of the primary treatment phase (Part 1), and every 8 weeks in the extension phase (Part 2).

[0247] Safety variables evaluated included: AEs (including serious AEs (SAEs)); clinical laboratory (chemistry, hematology, and urinalysis) results; vital signs (including growth); physical examination; electrocardiogram (ECG) test results; and immunogenicity test results (anti-PEG IgG, anti-PEG IgM, anti-PAL IgG, anti-PAL IgM, TAb, NAb, complement C3 and C4, and anti-PEG-valiase IgE [hypersensitivity visits only]).

[0248] Safety assessments in Part 1 will be conducted weekly for the first 8 weeks, then at 4-weekly visits until Week 73, with visits every 8 weeks during Part 2. Telephone assessments will be conducted during weeks when subjects do not have scheduled visits to answer dosing questions and review AEs and concomitant medications. Immunogenicity studies will be conducted on Days 1, 4, 8, 12, 16, 20, 24, and every 8 weeks thereafter in Part 1 and every 8 weeks in Part 2.

[0249] Secondary efficacy evaluations will be conducted to assess the effect of treatment with pegvaliase on neurocognitive outcomes in adolescent subjects with PKU. Neurocognitive assessments using the Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS) Inattention subscore and the Behavior Rating Inventory of Executive Function (BRIEF) will be conducted on Day 1 (baseline, before study drug) and every 12 weeks.

[0250] To assess dietary protein intake from untreated foods in adolescent subjects with PKU following treatment with pegvaliase, changes in protein intake from medical foods and / or untreated foods following treatment with the investigational drug will be examined.

[0251] To assess the pharmacokinetics (PK) of pegvaliase in adolescent subjects with PKU, plasma samples for trough PK samples will be collected on Day 1 and every 4 weeks for the first 24 weeks, followed by sampling every 8 weeks during Part 1. Focused PK sampling will be performed at Week 73 in all subjects. Samples will be collected pre-dose, and 2, 4, 8, 12, and 24 hours post-dose. A 24-hour sample will be collected prior to the next daily dose. Trough PK samples will be collected every 8 weeks during Part 2.

[0252] Tertiary efficacy evaluations will be performed to investigate the biochemical, molecular, and cellular aspects of PKU. Blood and urine samples will be collected to evaluate the biochemical, molecular, and cellular aspects of PKU and to develop the assays used for these evaluations.

[0253] (Comprehensive test design) (Subject Eligibility (Cohorts A and B)) To evaluate the safety and efficacy of PEGvaliase self-administered daily by adolescent subjects (ages 12-17 years, inclusive) with PKU, two cohorts in the study will be enrolled, dosed, and evaluated in parallel. Cohort A will include subjects aged 16-17 years at screening, and Cohort B will include subjects aged 12-15 years at screening.

[0254] Subjects who meet any of the following criteria are ineligible for study participation: previous treatment with PEGvaliase; use of any investigational drug or investigational device within 30 days prior to screening / observation, or need for any investigational medication before completion of all planned study evaluations; use of any medication intended to treat PKU, such as use of large neutral amino acids, within 14 days prior to administration of study drug on Day 1; use or planned use of injectable drugs containing polyethylene glycol (PEG; other than PEGvaliase), such as medroxyprogesterone injections, within 3 months prior to screening / observation and while participating in the study; positive test for HIV antibodies, hepatitis B surface antigen, or hepatitis C antibodies; history of organ transplant or chronic immunosuppressant therapy; substance abuse in the past 12 months (see the American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders history of pregnancy or breastfeeding at screening / observation or planning to become pregnant (own or partner) or breastfeed at any time during the study; intercurrent disease or illness that may interfere with study participation or safety (e.g., history or presence of clinically significant cardiovascular, pulmonary, hepatic, renal, hematological, gastrointestinal, endocrine, immune, dermatological, neurological, oncological, or psychiatric disease); major surgical procedure planned during the study; any condition that, in the opinion of the investigator, places the subject at high risk for poor treatment compliance or premature withdrawal from the study; alanine aminotransferase (ALT) concentration >2× upper limit of normal (ULN); and creatinine >1.5×ULN.

[0255] (Design of Cohort B (Overview)) The Cohort B design is an open-label, two-arm, randomized controlled evaluation of 27 subjects aged 12-15 years at US and EU study sites, with dietary restriction alone as the control arm. At enrollment, Cohort B subjects will be randomized 2:1 to the active arm or control arm, with 18 subjects receiving pegvaliase daily and 9 subjects managing their PKU with dietary restriction alone. Treatment assignment will be stratified by baseline blood Phe (average of last assessment) of ≤1000 μmol / L or >1000 μmol / L. The primary efficacy outcome measure will be the change in blood Phe from baseline at week 73. Cohort B subjects in the active and control arms will follow the same visit schedule and undergo the same assessments throughout the 73-week first-line treatment phase (Part 1), except that control subjects will not receive pegvaliase and will not undergo PK blood draws. Details of the induction / titration / maintenance (I / T / M) dosing regimen for active subjects in Cohort B are as described above for Cohort A. The timing of Part 1 assessments for active and control subjects in Cohort B are provided in Figures 3 and 5, respectively.

[0256] After week 73, 18 subjects in the active treatment arm of Cohort B will continue to receive open-label PEGValiase for up to an additional 80 weeks in the extension phase (Part 2). After week 73, 9 subjects in the control arm of Cohort B will begin treatment with PEGValiase and will follow a similar schedule of dosing, study visits, and assessments from week 74 through week 146 as subjects in the active arm of Part B follow from week 1 through week 73 during the primary treatment phase (Part 1). The timing of Part 2 assessments for active and control subjects in Cohort B are provided in Figures 4 and 6, respectively.

[0257] (summary) The objectives of this study are to evaluate the safety and efficacy of PEGValiase and to characterize the PK of PEGValiase in adolescent subjects with PKU. Study details include:

[0258] (Condition / Disease) Phenylketonuria (PKU) is a rare autosomal recessive genetic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that leads to absent or deficient PAH enzyme activity and subsequent elevation of the amino acid phenylalanine (Phe) in the blood, known as hyperphenylalaninemia (HPA) (Vockley et al., Genet Med. 16(2):188-200 (2014); Mitchell et al., Genet Med. 13(8):697-707 (2011)). Lack of PAH leads to abnormally elevated concentrations of Phe, which are toxic to the brain. High Phe levels during infancy and early childhood cause severe cognitive neurochemical and developmental defects, and poorly controlled blood Phe levels in older children and adults are associated with learning disabilities, attention deficit hyperactivity disorder, behavioral problems, and psychiatric symptoms.

[0259] (Test hypothesis) The safety population consisted of all subjects who received at least one dose of study drug. The efficacy population consisted of all subjects who received at least one dose of study drug during the study and had a post-treatment blood Phe measurement. Categorical data are presented using number of subjects and percentages. Continuous variables are presented using number of subjects, mean, standard deviation (SD), median, minimum, and maximum.

[0260] For Cohort A, the primary analysis for efficacy endpoints is descriptive. Additionally, 95% confidence intervals for the mean change in blood Phe at Week 73 are presented and compared to 250 μmol / L using an analysis of covariance (ANCOVA) model with baseline blood Phe as a covariate. For Cohort B, the primary analysis for efficacy (blood Phe at Week 73) is an ANCOVA model with baseline blood Phe as a covariate and treatment group as a factor. LS means and 95% confidence intervals are calculated for the treatment difference between PEGvaliase and diet alone.

[0261] Safety analyses will be performed on the safety population. AEs will be coded using the most recent version of the Medical Dictionary for Regulatory Activities (MedDRA). Incidences of treatment-emergent AEs, SAEs, and AEs of special interest will be summarized by MedDRA organ system class, preferred term, relationship to study drug, and severity. Subjects presenting with AEs leading to discontinuation of study drug, interruption of dosing, or reduction in dose level will be tabulated. A subject-specific listing of all AEs will be provided.

[0262] Clinical laboratory data will be summarized descriptively at baseline and post-baseline visits. Shift tables will be generated to summarize the change in Common Terminology Criteria for Adverse Events (CTCAE) grade from baseline to worst post-baseline value. Clinically significant laboratory abnormalities reported as AEs will be summarized. Descriptive statistics will also be provided for vital signs, physical examination results, ECG test results, and immunogenicity test results. Details of the statistical methods will be provided in the Statistical Analysis Plan (SAP).

[0263] (Exam period) The study duration will be up to 157 weeks for subjects in Cohort A randomized to treatment with active drug and for subjects in Cohort B. This will include a 4-week screening / run-out period in Parts 1 and 2 and up to 153 weeks of treatment with pegvaliase. The study duration will be 150 weeks for subjects in Cohort B randomized to the diet-only control arm. This will include a 4-week screening / run-out period, 73 weeks of management of PKU with diet alone following the Part 1 assessment schedule, and 73 weeks of treatment with pegvaliase from Weeks 74 to 146 following the same Part 1 assessment schedule.

[0264] (Duration of treatment (treatment defined as subcutaneous PEGylase)) The treatment duration will be up to 153 weeks for subjects in Cohort A and those randomized to pegvaliase in Cohort B. The treatment duration will be 73 weeks for subjects in Cohort B randomized to the diet-only control arm.

[0265] (Health Measurement / Observation) Decreased blood Phe levels.

[0266] (Frequency of visits) For subjects in the active (PEGValliase) arm of Cohort A and Cohort B, visits in Part 1 will be weekly for the first 8 weeks, then every 4 weeks until week 73, and every 8 weeks during Part 2. For subjects in the control (diet only) arm of Cohort B, visits in Part 1 will be weekly for the first 3 weeks, then every 4 weeks until week 73. After week 73, these subjects will begin treatment with PEGValliase while repeating the visit schedule for Part 1, i.e., weekly for 8 weeks, then every 4 weeks until week 146.

[0267] (Number of targets) Approximately 25 subjects will be enrolled in Cohort A. In subjects (ages 16-57 years) who completed 17 months of treatment in a previous pegvaliase trial utilizing the I / T / M dosing regimen, the mean (standard deviation [SD]) decrease in blood Phe concentration from treatment-naïve baseline at month 17 was 640 μmol / L (570 μmol / L). Assuming a similar treatment effect in adolescents aged 16-17 years, including the 25 subjects, there will be >90% power to detect a decrease in blood Phe concentration from treatment-naïve baseline at month 17 that is statistically significantly different from 250 μmol / L. Analyses will be based on two-tailed one-sample T-tests for blood Phe change from baseline and a significance level of 0.05.

[0268] Approximately 27 subjects will be enrolled in Cohort B and randomized in a 2:1 ratio to either the PEGylase or diet alone group. Assuming a mean (SD) reduction in Phe at week 73 of 640 (570) μmol / L in the PEGylase group and 100 (250) μmol / L in the diet alone group, a total sample size of 27 subjects will provide >90% power to detect a treatment difference based on a two-sample T-test with unequal variances and a significance level of 0.05 (2-sided).

[0269] (Treatment group and treatment period) The study drug is PEGValiase (formerly known as BMN 165; commercial name Palynziq; recombinant Anabaena variabilis phenylalanine ammonia-lyase-PEG [rAvPAL-PEG]), which will be provided to subjects in prefilled syringes (PFS) for self-administration. PEGValiase will be provided in the PFS in three dose strengths: 2.5 mg (0.5 mL of 5 mg / mL protein concentration), 10 mg (0.5 mL of 20 mg / mL protein concentration), and 20 mg (1.0 mL of 20 mg / mL protein concentration).

[0270] Subjects receive PEGValiase at a concentration of 5.0 or 20.0 mg / mL. PEGValiase is administered subcutaneously at dose levels ranging from 2.5 to 60 mg. The minimum dose of PEGValiase is 2.5 mg / week. The maximum tolerated daily dose of PEGValiase is 60 mg / day (for a maximum weekly dose of 420 mg). The time between each dose adjustment step may be increased depending on AEs. Reductions in dose levels due to AEs or hypophenylalaninemia (blood Phe levels of <30 μmol / L) may be performed at any time during the study.

[0271] The duration of the first treatment phase (part 1) is 73 weeks, during which treatment with pegvaliase is initiated using the I / T / M dosing regimen. The recommended dosing schedule for all subjects receiving pegvaliase in either cohort A or B is shown in Table 3. Induction consists of a 4-week period during which subjects receive pegvaliase subcutaneously at a fixed dose of 2.5 mg / week with PFS, during which the dosing regimen is not altered according to blood Phe levels. Once subjects have completed induction, they will be titrated up to a dose of 10 mg / day. Dosing frequency will be gradually increased to daily (7 days / week) during the titration phase. During the maintenance phase, doses will be increased to 20 mg / day, 40 mg / day, and 60 mg / day depending on individual blood Phe-lowering efficacy (at the investigator's discretion). Dose should be increased to 40 mg / day if blood Phe is >360 μmol / L after 24 weeks of dosing at 20 mg / day, and to 60 mg / day if blood Phe is >360 μmol / L after 16 weeks of dosing at 40 mg / day. Target blood Phe levels should be based on individual need, with a minimum target blood Phe of <600 μmol / L.

[0272] After completion of Part 1, to evaluate the long-term safety and efficacy of PEGValiase in adolescent subjects with PKU, subjects will enter an extension phase (Part 2), during which subjects will continue to receive PEGValiase at up to 60 mg / day for up to an additional 80 weeks. Table 3: Study Drug Dose Adjustment Schedule [Table 4] a Dose adjustments / escalations may be delayed depending on subject tolerance. b All subjects will begin maintenance at 20 mg per day. Doses will be increased to 40 mg / day and / or 60 mg / day only if needed.

[0273] After enrollment, the subject (and the subject's designated caregiver) will be trained to administer the study drug at home. At a minimum, the first three study drug doses (weeks 1, 2, and 3) will be administered by the subject (or caregiver) at the clinic under the supervision of clinic personnel. To be allowed to administer the study drug at home, the subject (or caregiver) must demonstrate the ability to inject the study drug. During Part 1 of treatment (i.e., through week 73), a competent adult (trained adult observer) must also be present during and for a minimum of 1 hour after administration of the study drug. Administration of the study drug may only be performed if this adult observer is present. The subject and the trained adult observer who will observe the subject during administration of the study drug will be provided with information and extensive training on possible allergic reactions, how to recognize the severity of the reaction, and instructions on what to do if a reaction occurs.

[0274] Requiring a trained adult observer for self-administered doses beyond the first 73 weeks of treatment is considered optional for individual subjects based on the investigator's clinical judgment. For example, a trained adult observer may be considered for subjects with intellectual disability or for subjects with prior experience of anaphylaxis per National Institute of Allergy and Infectious Disease / Food Allergy and Anaphylaxis Network (NIAID / FAAN) criteria.

[0275] Subjects will be provided with two epinephrine syringes and instructed to carry at least one epinephrine syringe at all times. Subjects and trained adult observers will be trained to recognize the signs and symptoms of anaphylaxis or acute systemic hypersensitivity reactions (ASHRs) and to call for emergency medical assistance and administer an epinephrine syringe if these reactions occur. Throughout the study, each subject will be contacted by study site personnel to monitor for self-administration issues and adverse events (AEs).

[0276] During the induction and titration phases, all subjects receiving PEG-valiase will be premedicated with an H1 antagonist, an H2 antagonist, and an antipyretic approximately 2-3 hours prior to each dose of study drug. At the investigator's discretion, premedication may be considered during the maintenance phase. Premedication may also be performed based on clinical judgment approximately 2-3 hours prior to study drug upon reintroduction of study drug, upon resolution of AEs, after a ≥4 day dosing interruption, and for 1 week for dose escalation in the extension phase. Subjects may also be premedicated at any time during the study at the investigator's discretion. This premedication regimen has been successfully implemented in a Phase 3 adult clinical program. Subjects will be provided with a logbook to record the date and time of study drug injection, injection site, use of premedication, and suspected AEs.

[0277] If ≥ 4 doses are not administered for reasons other than safety-related issues, the investigator should consult with and obtain approval from the medical monitor before the subject resumes study drug.

[0278] (Dietary monitoring) Subjects will be asked to maintain a stable dietary protein intake from medical foods and / or untreated foods throughout the 73-week I / T / M phase (Part 1). To ensure efficacy and safety endpoints can be attributed to study treatment and not to changes in dietary protein intake, subjects' ability to maintain a stable protein intake is essential to the success of the study. Subjects will be required to maintain dietary protein intake levels consistent with baseline levels throughout the study, with a consistent diet defined as intact protein change <10% from baseline and medical food protein change <10% from baseline.

[0279] A dietician under the supervision of the investigator will be required to monitor and manage the subjects' diet throughout the study. Subjects will be provided with a 3-day diary in which all dietary protein intake (including medical and / or untreated foods) must be recorded for 3 consecutive days immediately prior to each scheduled visit for review by the dietician. All subjects will be offered the option of tyrosine supplementation (500 mg, 3 times per day with meals) at the investigator's discretion.

[0280] During the screening / run-out phase, two 3-day food diary assessments will be conducted, 2-4 weeks apart. Subjects will be instructed not to alter their dietary protein intake (medical food and / or untreated food) during the screening / run-out and primary treatment phases (Part 1) of the study. However, if blood Phe levels fall to <30 μmol / L and are confirmed on recheck (which will occur within approximately 4 weeks), the subject's diet should be modified and the investigational drug dose level reduced.

[0281] Once subjects enter the extension phase (Part 2), dietary protein intake may be modified if blood Phe concentrations remain ≦360 μmol / L for a minimum of 4 weeks. Subjects with blood Phe measurements ≦360 μmol / L during Part 2 may have their dietary protein intake adjusted based on the individual subject's response to PEG-valiase and guidance from the investigator or designee.

[0282] If the subject's dose level is reduced, a reduction in pegvaliase from 60 mg / day to 40 mg / day or 40 mg / day to 20 mg / day is recommended. Intermediate dose reductions may be permitted after discussion with the medical monitor. Dose reductions may be made over the phone or in the clinic. If diet is modified, it is recommended that intact protein be increased in 10 g increments unless the subject is already consuming age-appropriate protein levels (World Health Organization, Food and Agriculture Organization of the United Nations, 2007). If the dietician determines that essential amino acids have reached age-appropriate levels, the medical food may be discontinued.

[0283] (pregnancy) Because the risks of taking investigational product during pregnancy and lactation are unknown, subjects may not take the investigational product if they are trying to become pregnant, are pregnant, or are lactating. As outlined in the Inclusion Criteria section of this protocol, beginning on Day 28, sexually active subjects must use two acceptable methods of contraception. Subjects who are confirmed to be pregnant by serum pregnancy test and who are temporarily weaned from investigational product are not required to perform a scheduled urine pregnancy test.

[0284] (Target Cancellation) If the investigational product is discontinued prior to study completion, the investigator will ask the subject to remain in the study and continue study visits and evaluations. Subjects who discontinue investigational product early should continue to undergo study evaluations for 30 days after discontinuation, provided that such continued participation will not adversely affect the subject's health, safety, and well-being as determined by the investigator.

[0285] (Safety Management Plan) An independent Data Monitoring Committee (DMC) will monitor the safety of study subjects.

[0286] (Response to hypersensitivity events) Subjects will be evaluated for safety throughout the study and trained to recognize possible hypersensitivity AEs (HAEs), such as anaphylaxis or acute systemic hypersensitivity reactions (per NIAID / FAAN criteria), and how to respond. Subjects will be instructed to contact the investigator regarding any suspected HAEs. After telephone evaluation, the investigator may request further evaluation in the clinic. If a hypersensitivity reaction occurs (e.g., injection site reaction, rash, arthralgia, pruritus), subjects may be advised to premedicate with H1 antagonists, H2 antagonists, and antipyretics (e.g., acetaminophen) approximately 2-3 hours before the subsequent dose of study drug. If a nonsteroidal anti-inflammatory drug (NSAID) is administered as a premedication, it should be given with food.

[0287] If a subject develops a severe or dermatologically significant skin reaction (or a reaction that may possibly be vasculitis), the subject should be referred to a dermatologist. A skin biopsy may be considered as part of the evaluation of any such condition.

[0288] The occurrence of HAE due to study drug administration is anticipated. In response to a suspicion of HAE, dosing of the study drug may be modified or discontinued depending on the severity of the event and the suspected causality of the study drug. Individual AEs by Medical Dictionary for Clinical Practice (MedDRA) Preferred Term that qualify as HAEs are defined consistent with the definition of a "hypersensitivity reaction" adverse drug reaction (ADR). The severity of AEs (preferred terms) related to HAE are graded according to the National Cancer Institute - Common Terminology Criteria for Adverse Events (NCI-CTCAE) criteria.

[0289] Individual Stopping Criteria Assessment for Adverse Events During Treatment with PEGylalase Subjects who experience an anaphylaxis-related episode suspected to be related to the study drug and meeting Brown's criteria for severe symptoms (Grade 3), in the opinion of the investigator and / or sponsor's medical monitor, will be permanently discontinued from the study drug.

[0290] (Medications in response to hypersensitivity events) Dosing for HAE depends on the NCI-CTCAE grade and relationship to the suspected investigational drug. Dosing instructions are provided in Table 4 (regardless of prior occurrence). (Table 4: Medication instructions for hypersensitivity adverse events) [Table 5] AE, adverse event; CTCAE, Common Terminology Criteria for Adverse Events, version 5.0; HRV, hypersensitivity reaction visit; NCI, National Cancer Institute. a NCI-CTCAE grade determinations will be made by the investigator and may be performed either by telephone or in person. b The investigator will instruct the subject to remain on the study drug dose at the time of AE onset until improvement or resolution to Grade 1 (as assessed by the investigator in clinic or by telephone). c The study drug dose may be reduced or discontinued, if necessary, at the discretion of the investigator. d If a subject has an NCI-CTCAE Grade ≧3 hypersensitivity AE suspected to be related to the study drug and meets Brown's criteria for severe disease (Grade 3) in the judgment of the Investigator and / or Sponsor's Medical Monitor, the subject will be permanently discontinued from the study drug. eIf the investigator determines that a hypersensitivity reaction of NCI-CTCAE grade ≧3 is related to dosing with the study drug, the subject will be asked to return to the clinic within 24 hours of onset of the event for evaluation, including laboratory tests (chemistry, hematology, urinalysis, anti-PEGylation IgE [sample collection should be performed 8-24 hours after onset of the event and prior to the next dose of study drug], urinary albumin / creatinine ratio, CRP, C3, and C4).

[0291] Once the HAE (other than anaphylaxis) has improved or resolved to Grade 1, the study drug dose may be increased, maintained, or decreased at the investigator's discretion. If decreased, the reduction should be from 60 to 40 mg / day, 40 to 20 mg / day, or 20 to 10 mg / day, whichever is appropriate.

[0292] (Response to anaphylaxis or acute systemic hypersensitivity reactions) If a subject experiences anaphylaxis or an acute systemic hypersensitivity reaction, the subject will be instructed to receive an epinephrine injection, seek immediate medical attention, and notify the investigator.

[0293] If the investigator suspects that the AE is anaphylaxis as defined by NIAID / FAAN, the subject will be evaluated in the clinic and the sponsor's medical monitor should be notified immediately. Laboratory evaluation for suspected anaphylactic event will be performed prior to the next study drug administration (if applicable) and will include anti-PEG-variase IgE (for optimal results, sample collection should occur 8-24 hours after onset of the event). If the investigator determines that it is safe for a subject to resume dosing with the investigational product after resolution of anaphylaxis at any time during the study, the following steps are required: at least the first dose administered after resolution of anaphylaxis will be given in a clinic with easily accessible equipment for emergency resuscitation (including epinephrine); regardless of the length of the interruption, the subject must be premedicated with an H1 antagonist, an H2 antagonist, and an antipyretic (e.g., acetaminophen) approximately 2-3 hours before each dose of investigational product for at least 1 week upon resumption of dosing; regardless of the length of the interruption, a trained adult observer must observe the subject during and for a minimum of 1 hour after administration of the investigational product for at least 1 week after resumption of dosing; and investigational product may only be administered if this person is present.

[0294] (Study Stopping Criteria Assessment for Adverse Events During Treatment with the Investigational Drug) If an anaphylactic or acute systemic hypersensitivity reaction that meets the Brown criteria for severity (Grade 3; Table 4) occurs, the DRB chair and / or committee will be notified and will advise the sponsor on possible modifications to the study conduct. Clinically significant hypersensitivity according to the Brown criteria is defined as significant hypoxia, hypotension, or neurological impairment that was life-threatening or required treatment to prevent a life-threatening event: cyanosis or SpO2 ≤ 92%; hypotension with SBP < 90 mmHg; neurological changes (e.g., confusion, loss of consciousness, collapse, and incontinence).

[0295] Numerous modifications and variations in this disclosure, such as those described in the above examples, are expected to occur to those skilled in the art. As a result, only such limitations as appear in the appended claims should be imposed on this disclosure.

Claims

1. A pharmaceutical composition for use in a method for lowering blood phenylalanine levels in a subject, the method comprising administering to the subject a weekly dose of a formulation comprising an AvPAL variant, the subject being between about 12 and about 18 years of age, and the weekly dose being administered for greater than about 50 weeks, the AvPAL variant comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

2. 2. The pharmaceutical composition of claim 1, wherein said method comprises administering said weekly dose for more than about 60 weeks, more than about 70 weeks, more than about 80 weeks, more than about 90 weeks, more than about 100 weeks, more than about 110 weeks, more than about 120 weeks, more than about 130 weeks, more than about 140 weeks, more than about 150 weeks, more than about 160 weeks, more than about 170 weeks, more than about 180 weeks, more than about 190 weeks, more than about 200 weeks, more than about 210 weeks, more than about 220 weeks, more than about 230 weeks, more than about 240 weeks, or more than about 250 weeks.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 0.1 mg per week to about 1 mg per week.

4. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 1 mg per week to about 2 mg per week.

5. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 2 mg per week to about 10 mg per week.

6. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 10 mg per week to about 20 mg per week.

7. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 20 mg per week to about 40 mg per week.

8. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 40 mg per week to about 70 mg per week.

9. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 70 mg per week to about 140 mg per week.

10. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 140 mg per week to about 280 mg per week.

11. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 280 mg per week to about 420 mg per week.

12. 3. The pharmaceutical composition of claim 1 or 2, wherein the dosage ranges from about 420 mg per week to about 840 mg per week.

13. The pharmaceutical composition of claim 1 , wherein the method comprises administering the AvPAL variant once a week.

14. The pharmaceutical composition of claim 1 , wherein the method comprises administering the AvPAL variant twice weekly.

15. 2. The pharmaceutical composition of claim 1, wherein the method comprises administering the AvPAL variant four times per week.

16. 2. The pharmaceutical composition of claim 1, wherein the method comprises administering the AvPAL variant 7 times per week.

17. 2. The pharmaceutical composition of claim 1, wherein the AvPAL variant is administered 14 times per week.

18. The pharmaceutical composition of claim 1 , wherein the method comprises administering the AvPAL variant daily.

19. The method further comprising: a. administering to said subject said AvPAL variant at an induction dosage ranging from about 0.1 mg per week to about 10 mg per week; followed by b. administering to the subject the AvPAL variant in a titrated dosage ranging from about 1 mg per week to about 200 mg per week; followed by c. administering to the subject the AvPAL variant at a maintenance dosage ranging from about 20 mg per week to about 840 mg per week.

2. The pharmaceutical composition of claim 1, comprising:

20. 20. The pharmaceutical composition of claim 19, wherein the induction dosage is about 2.5 mg per week.

21. 21. The pharmaceutical composition of claim 19 or 20, wherein the titration dosage ranges from about 5 mg per week to about 70 mg per week.

22. 21. The pharmaceutical composition of claim 19 or 20, wherein the maintenance dosage ranges from about 140 mg per week to about 420 mg per week.

23. 21. The pharmaceutical composition of claim 19 or 20, wherein in said method, the induction dose is administered for a period of about 2 weeks to about 6 weeks, the titration dose is administered for a period of about 3 weeks to about 8 weeks, and the maintenance dose is administered for a period of about 50 weeks to about 80 weeks.

24. 24. The pharmaceutical composition of claim 23, wherein said method wherein said induction dose is administered for a period of about 4 weeks, said titration dose is administered for a period of about 5 weeks, and said maintenance dose is administered for a period of about 56 to 64 weeks.

25. 24. The pharmaceutical composition of claim 23, wherein the maintenance dosage amounts consist of a first maintenance dosage amount of about 70 mg per week to about 280 mg per week, a second maintenance dosage amount of about 140 mg per week to about 560 mg per week, and a third maintenance dosage amount of about 210 mg per week to about 840 mg per week.

26. 26. The pharmaceutical composition of claim 25, wherein said method wherein the first maintenance dosage is administered for a period of about 16 weeks to about 24 weeks, the second maintenance dosage is administered for a period of about 16 weeks, and the third maintenance dosage is administered for a period of about 24 weeks.

27. 20. The pharmaceutical composition of claim 19, wherein the method further comprises administering to the subject an extended dosage of the AvPAL variant in the range of about 20 mg per week to about 840 mg per week after administration of the maintenance dosage.

28. 28. The pharmaceutical composition of claim 27, wherein said method wherein said extended dosage is administered for a period of about 40 weeks to about 120 weeks.

29. 30. The pharmaceutical composition of claim 28, wherein said method comprises administering said induction dose for a period of about 4 weeks, said titration dose for a period of about 5 weeks, said maintenance dose for a period of about 64 weeks, and said extension dose for a period of about 80 weeks.

30. 30. The pharmaceutical composition of claim 19 or 27, wherein the method further comprises assessing the blood phenylalanine concentration prior to administering the loading dose.

31. 30. The pharmaceutical composition of claim 19 or 27, wherein the method further comprises evaluating the blood phenylalanine concentration after administration of one or more induction doses, titration doses, maintenance doses, and / or extension doses.

32. 32. The pharmaceutical composition of claim 31, wherein the method further comprises adjusting the dosage based on the blood phenylalanine concentration.

33. 33. The pharmaceutical composition of claim 32, wherein said dosage is adjusted to achieve a blood phenylalanine concentration of less than about 600 μM.

34. 33. The pharmaceutical composition of claim 32, wherein said dosage is adjusted to achieve a blood phenylalanine concentration of less than about 360 μM.

35. 34. The pharmaceutical composition of claim 33, wherein the method comprises increasing the maintenance dosage when the blood phenylalanine concentration is greater than about 360 μM.

36. 30. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the subject is suffering from phenylketonuria (PKU).

37. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the subject is about 12 to about 15 years old.

38. 28. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the subject is about 16 to about 17 years old.

39. 30. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:

2.

40. 30. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:

3.

41. 30. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the AvPAL variant comprises the amino acid sequence of SEQ ID NO:

4.

42. 30. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the AvPAL variant is pegylated.

43. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of at least 1.6 polyethylene glycol per lysine residue of AvPAL variant.

44. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of at least 2.4 polyethylene glycol per lysine residue of AvPAL variant.

45. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of three polyethylene glycol per lysine residue of AvPAL variant.

46. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 5 polyethylene glycol per lysine residue of AvPAL variant.

47. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of six polyethylene glycol per lysine residue of AvPAL variant.

48. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 7 polyethylene glycol per lysine residue of AvPAL variant.

49. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 8 polyethylene glycol per lysine residue of AvPAL variant.

50. 43. The pharmaceutical composition of claim 42, wherein said pegylation is accomplished by reacting said AvPAL variant with NHS-activated polyethylene glycol in a ratio of 9 polyethylene glycol per lysine residue of AvPAL variant.

51. 30. The pharmaceutical composition of any one of claims 1, 19, and 27, wherein the AvPAL variant is administered in a formulation comprising a pharma- ceutically acceptable carrier that comprises a stabilizing agent.

52. 52. The pharmaceutical composition of claim 51, wherein the stabilizer is L-phenylalanine or a structural analog thereof.

53. 53. The pharmaceutical composition of claim 52, wherein the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid, and benzoic acid.

54. 54. The pharmaceutical composition of claim 53, wherein the stabilizer is trans-cinnamic acid.

55. 55. The pharmaceutical composition of claim 54, wherein the formulation further comprises sodium chloride, and tromethamine and tromethamine hydrochloride.

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