Patent Publication Number: US-2015086530-A1

Title: Dosing regimens for the treatment of pompe disease

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application Ser. No. 61/642,311 filed May 3, 2012, U.S. Provisional Application Ser. No. 61/664,011 filed Jun. 25, 2012, U.S. Provisional Application Ser. No. 61/697,179 filed Sep. 5, 2012, U.S. Provisional Application Ser. No. 61/749,132 filed Jan. 4, 2013, and U.S. Provisional Application Ser. No. 61/749,234 filed Jan. 4, 2013, to each of which priority is claimed and each of which are incorporated herein by reference in their entireties. 
    
    
     FIELD OF THE APPLICATION 
     The present application provides a dosing regimen and administration schedule for the use of 1-deoxynojirimycin and enzyme replacement therapy for the treatment of Pompe disease. 
     BACKGROUND 
     Mutations in the lysosomal enzyme acid α-glucosidase (GAA) alter lysosomal glycogen catabolism and lead to Pompe disease, also referred to as glycogen storage disease type II or acid maltase deficiency. GAA normally hydrolyzes glycogen at α-1,4 and α-1,6 linkages to yield glucose. Mutations in the GAA gene result in a deficiency or absence of GAA activity, which leads to an accumulation of glycogen. The glycogen accumulation is thought to lead to progressive muscle myopathy throughout the body, affecting various body tissues, particularly the heart, skeletal muscles, liver, and nervous system. In some cases of Pompe disease, reduced levels of the 110-kDa GAA precursor protein are observed, while in other cases normal levels of 110-kDa precursor protein are synthesized but not processed into the mature, properly glycosylated 76- and 70-kDa GAA forms. 
     Pompe disease has been historically divided into three major phenotypic expressions (infantile-, juvenile- and adult-onset). However, it is now currently accepted that the disease is a spectrum of phenotypes, ranging from the more severe early-onset form to the less severe late-onset form. The disorder is clinically heterogeneous in age of onset, extent of organ involvement, and rate of progression. The early-onset form of the disease is the most severe, and progresses most rapidly, with accumulation of glycogen most prevalent in cardiac muscle, skeletal muscle, and hepatic tissue. This form of the disease typically is characterized by musculoskeletal, pulmonary, gastrointestinal, and cardiac symptoms. Death due to cardiorespiratory failure usually occurs between 1 and 2 years of age. The late-onset form of the disease usually begins between childhood and adulthood and has a slower rate of progression, usually without cardiac involvement. The phenotype is characterized by musculoskeletal and pulmonary symptoms that leads to progressive weakness and respiratory insufficiency. The symptoms tend to be less severe, and glycogen accumulation less extensive, in the late-onset than in the early-onset form of the disease, resulting in longer survival. Death usually results due to cardiorespiratory failure. 
     Current treatment of Pompe disease involves symptomatic treatment of the cardiac and respiratory symptoms. There is no approved treatment for the underlying genetic defect. Use of replacement GAA, alglucosidase alfa (Myozyme® (Genzyme Corporation) and Lumizyme® (Genzyme Corporation)) is approved by the F.D.A. in the United States. However, clinical evaluations using enzyme replacement therapy (ERT) to replace defective GAA in infantile Pompe patients was only moderately successful in improving cardiac and skeletal function (Klinge et al.,  Neuropediatrics.  2005; 36(1): 6-11). Recombinant GAA was shown to be more effective in resolving the cardiomyopathy than the skeletal muscle myopathy (Raben et al.,  Mol Ther.  2005; 11(1): 48-56), largely because recombinant enzyme cannot penetrate connective tissue. A method for treating Pompe disease using recombinant GAA is specifically described in U.S. Pat. No. 6,537,785 to Canfield. One of the main complications with ERT is the attainment and maintenance of therapeutically effective amounts of enzyme due to rapid degradation of the infused enzyme. 
     1-deoxynojirimycin and its salt, 1-deoxynojirimycin hydrochloride, acts as a pharmacological chaperone for mutant GAA by selectively binding to the enzyme, thereby increasing its stability and helping the enzyme fold into its correct three-dimensional shape. This stabilization of GAA allows the cell&#39;s quality control mechanisms to recognize the enzyme as properly folded so that trafficking of the enzyme to the lysosome is increased, allowing it to carry out its intended biological function, the metabolism of glycogen. As a result of restoring the proper trafficking of GAA from the ER to the lysosome, 1-deoxynojirimycin hydrochloride also reduces the accumulation of misfolded protein in the ER, which can alleviate stress on cells and some inflammatory-like responses that can be contributing factors in Pompe disease. Multiple in vitro and in vivo preclinical studies, as well as clinical studies, of 1-deoxynojirimycin hydrochloride have been conducted. 1-deoxynojirimycin hydrochloride has been shown to increase the amount of intracellular GAA protein and to enhance transport of mutant enzyme to the lysosome. 
     SUMMARY 
     The present application provides a dosing regimen and administration schedule for the use of 1-deoxynojirimycin and enzyme replacement therapy for the treatment of Pompe disease. In certain embodiments, the present application provides a dosing regimen and administration schedule for the use of 1-deoxynojirimycin hydrochloride and alglucosidase alfa for the treatment of Pompe disease. 
     In one embodiment, the method includes administering from about 25 mg to about 1000 mg of 1-deoxynojirimycin and an effective amount of GAA enzyme replacement therapy to a patient in need thereof. The 1-deoxynojirimycin may be administered before, after, or simultaneously with the GAA enzyme replacement therapy. In one embodiment, the patient fasts for a period of time beginning about 0.5 to about 4 hours prior to and ending about 0.5 to about 4 hours following administration of 1-deoxynojirimycin. In a further embodiment, the patient fasts for at least about 2 hours prior to and at least about 2 hours following administration of 1-deoxynojirimycin 
     In another embodiment, the 1-deoxynojirimycin is administered simultaneously with to about 4 hours prior to the administration of the GAA enzyme replacement therapy (from T=−4 hours to T=0 hours). In a further embodiment, the 1-deoxynojirimycin is administered about 2 hours prior to the administration of the GAA enzyme replacement therapy. 
     In a particular embodiment, the 1-deoxynojirimycin is 1-deoxynojirimycin hydrochloride. In one embodiment, the GAA enzyme replacement therapy is rhGAA. In a further embodiment, the GAA enzyme replacement therapy is alglucosidase alfa. 
     In one embodiment, the 1-deoxynojirimycin is administered as an adjuvant to the GAA enzyme replacement therapy. In another embodiment, the 1-deoxynojirimycin and GAA enzyme replacement therapy are administered as a combination therapy. 
     In a particular embodiment, the amount of 1-deoxynojirimycin administered according to the above-described method is from about 50 mg to about 600 mg. In one embodiment, the amount of 1-deoxynojirimycin administered is selected from 50 mg, 100 mg, 250 mg and 600 mg. 
     In a particular embodiment, the 1-deoxynojirimycin is administered immediately before or at the same time as the administration of the GAA enzyme replacement therapy. In an alternate embodiment, the patient is administered a second dose of 1-deoxynojirimycin between the administration of the GAA enzyme replacement therapy and about 4 hours thereafter. 
     In certain embodiments, the 1-deoxynojirimycin is administered every 1 to 4 weeks to a patient who is also receiving GAA enzyme replacement therapy. In a further embodiment, the 1-deoxynojirimycin is administered every 12 to 16 days to a patient who is also receiving GAA enzyme replacement therapy. In certain embodiments, the GAA enzyme replacement therapy is administered every 14 days to the patient who is also administered 1-deoxynojirimycin as a combination of adjuvant therapy. 
     The present application also provides a kit for treating Pompe disease in a subject, the kit comprising from about 25 mg to about 1000 mg of 1-deoxynojirimycin and an effective amount of GAA enzyme replacement therapy. In certain embodiments, the amount of 1-deoxynojirimycin in the kit is selected from 50 mg, 100 mg, 250 mg and 600 mg. 
     The present application provides a dosing regimen and administration schedule for the use of 1-deoxynojirimycin (1-DNJ) derivatives and enzyme replacement therapy for the treatment of Pompe disease. 
     In one embodiment, the method includes administering from about 25 mg to about 1000 mg of a 1-deoxynojirimycin derivative and an effective amount of GAA enzyme replacement therapy to a patient in need thereof. The 1-deoxynojirimycin derivative may be administered before, after, or simultaneously with the GAA enzyme replacement therapy. In one embodiment, the patient fasts for a period of time beginning about 0.5 to about 4 hours prior to and ending about 0.5 to about 4 hours following administration of 1-deoxynojirimycin derivative. In a further embodiment, the patient fasts for at least about 2 hours prior to and at least about 2 hours following administration of 1-deoxynojirimycin derivative. 
     In another embodiment, the 1-deoxynojirimycin derivative is administered simultaneously with to about 4 hours prior to the administration of the GAA enzyme replacement therapy (from T=−4 hours to T=0 hours). In a further embodiment, the 1-deoxynojirimycin derivative is administered about 2 hours prior to the administration of the GAA enzyme replacement therapy. 
     In some embodiments, the 1-deoxynojirimycin derivative is (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, or miglustat, or N-Butyl DNJ. In one embodiment, the GAA enzyme replacement therapy is rhGAA. In a further embodiment, the GAA enzyme replacement therapy is alglucosidase alfa. 
     In one embodiment, the 1-deoxynojirimycin derivative is administered as an adjuvant to the GAA enzyme replacement therapy. In another embodiment, the 1-deoxynojirimycin derivative and GAA enzyme replacement therapy are administered as a combination therapy. 
     In a particular embodiment, the amount of 1-deoxynojirimycin derivative administered according to the above-described method is from about 50 mg to about 600 mg. In one embodiment, the amount of 1-deoxynojirimycin derivative administered is selected from 50 mg, 100 mg, 250 mg and 600 mg. 
     In a particular embodiment, the 1-deoxynojirimycin derivative is administered immediately before or at the same time as the administration of the GAA enzyme replacement therapy. In an alternate embodiment, the patient is administered a second dose of 1-deoxynojirimycin derivative between the administration of the GAA enzyme replacement therapy and about 4 hours thereafter. 
     In certain embodiments, the 1-deoxynojirimycin derivative is administered every 1 to 4 weeks to a patient who is also receiving GAA enzyme replacement therapy. In a further embodiment, the 1-deoxynojirimycin derivative is administered every 12 to 16 days to a patient who is also receiving GAA enzyme replacement therapy. In certain embodiments, the GAA enzyme replacement therapy is administered every 14 days to the patient who is also administered 1-deoxynojirimycin derivative as a combination of adjuvant therapy. 
     In further embodiments of the claimed method, the 1-DNJ derivative is selected from the group consisting of N-methyl-DNJ, N-butyl-DNJ, N-cyclopropylmethyl-DNJ, N-(2-(N,N-dimethylamido)ethyloxy-DNJ, N-4-t-butyloxycarbonyl-piperidnylmethyl-DNJ, N-2-R-tetrahydrofuranylmethyl-DNJ, N-2-R-tetrahydrofuranylmethyl-DNJ, N-(2-(2,2,2-trifluoroethoxy)ethyl-DNJ, N-2-methoxyethyl-DNJ, N-2-ethoxyethyl-DNJ, N-4-trifluoromethylbenzyl-DNJ, N-alpha-cyano-4-trifluoromethylbenzyl-DNJ, N-4-trifluoromethoxybenzyl-DNJ, N-4-n-pentoxybenzyl-DNJ, and N-4-n-butoxybenzyl-DNJ, or C1-nonyl DNJ. 
     The present application also provides a kit for treating Pompe disease in a subject, the kit comprising from about 25 mg to about 1000 mg of 1-deoxynojirimycin derivative and an effective amount of GAA enzyme replacement therapy. In certain embodiments, the amount of 1-deoxynojirimycin derivative in the kit is selected from 50 mg, 100 mg, 250 mg and 600 mg. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A-B  shows composite graphs of rhGAA plasma activity in the four Cohort 1 subjects by treatment, as described by Example 2.  FIG. 1A  shows rhGAA plasma activity following treatment with GAA ERT alone.  FIG. 1B  shows rhGAA plasma activity following treatment with co-administration of GAA ERT and 50 mg 1-deoxynojirimycin hydrochloride. 
         FIG. 2A-B  shows (A) the mean rhGAA plasma activity in the four Cohort 1 subjects by treatment, and (B) mean total rhGAA protein as measured by western blot in the four Cohort 1 subjects by treatment, as described by Example 2. 
         FIG. 3A-B  shows composite graphs of rhGAA plasma activity in the six Cohort 2 subjects by treatment, as described by Example 2.  FIG. 3A  shows rhGAA plasma activity following treatment with GAA ERT alone.  FIG. 3B  shows rhGAA plasma activity following treatment with co-administration of GAA ERT and 100 mg 1-deoxynojirimycin hydrochloride. 
         FIG. 4  shows the mean rhGAA plasma activity in the six Cohort 2 subjects by treatment. 
         FIG. 5  shows the mean (SD) plasma 1-DNJ-HCL concentration-time profiles of Cohort 1 and 2 subjects by treatment. 
         FIG. 6  shows the total plasma rhGAA protein concentration profiles of Cohort 1 and 2 subjects by treatment, as measured by Western blot. 
         FIG. 7  shows urine Hex 4 levels by study day of Cohort 1 and 2 subjects, which was collected as part of the safety data. 
         FIG. 8  shows serum CPK levels by study day of Cohort 1 and 2 subjects, which was collected as part of the safety data. 
         FIG. 9A-B  shows Composite Plasma rhGAA Activity by Treatment Period for cohort 3 subjects. 
         FIG. 10A-D  shows AT2220-010 Mean (SD) Plasma rhGAA Activity for cohorts 1-4. 
         FIG. 11  shows AUC Stick Plot for rhGAA Activity in Plasma for cohort 3 subjects. 
         FIG. 12A-B  shows rhGAA Activity in Muscle from biopsies taken on Days 3 or 7 of Periods 1 and 2, and an optional baseline biopsy at follow up, for cohort 3 subjects. 
         FIG. 13  shows a Composite Muscle rhGAA Activity Summary by Treatment Period for cohort 1-3 subjects. 
         FIG. 14A-B  shows rhGAA Activity in PBMCs (peripheral blood mononuclear cells): Day 3 composites by treatment for cohort 3 subjects. 
         FIG. 15A-B  shows rhGAA Activity in PBMCs: Day 7 composites by treatment for cohort 3 subjects. 
         FIG. 16A-B  shows a Composite of plasma AT2220 Concentrations and a semi-log plot for cohort 3 subjects. 
         FIG. 17A-B  shows a Composite of Plasma rhGAA Activity by Treatment Period for cohort 4 subjects. 
         FIG. 18  shows AUC Stick Plot for rhGAA Activity in Plasma for cohort 4 subjects. 
         FIG. 19  shows a Composite Muscle rhGAA Activity Summary by Treatment Period for cohorts 1-4. 
         FIG. 20  shows Thigh Muscle MRI before (top) and after (bottom) ERT. At one year, 9.8% decrease in muscle mass in the anterior thigh, 11% decrease in muscle mass in posterior thigh, and +8.9% increase in intramuscular fat accumulation (6.6% subcutaneous fat). 
         FIG. 21  shows a summary of rhGAA Enzyme Activity in Plasma Area Under Curve (AUC) for cohorts 1-4. 
         FIG. 22  shows a summary of GAA Enzyme Activity in Muscle at Day 3 for cohorts 2-4. 
         FIG. 23  shows that the skeletal muscle distribution and half-life of N-butyl-DNJ (AT2221) is similar to 1-DNJ (AT2220); Cmax AT2220: 120 uM; AT2221: 140 uM. Eight-week old wild type C57BL/6 mice where administered an oral dose of 100 mg/kg of 1-DNJ or N-butyl-DNJ. Plasma and tissue samples were taken at 0.5, 2, 4, 24, 48, 72, 96, 120, 144, &amp; 168 hours post administration and the presence of drug was analyzed. The concentration of drug in plasma is expressed as ng/ml. The concentration of drug in tissue samples is expressed as ng/g. 
         FIG. 24  shows that N-butyl-DNJ (AT2221) and 1-DNJ (AT2220) have a similar effect on Phramacokinetics of rhGAA. Eight week old GAA KO mice were administered rhGAA (10 mg/kg IV). Oral AT2220 or AT2221 (100 mg/kg) was administered 30 min prior to GAA (Myozyme); Plasma samples were taken at pre-dose, 0.08, 0.25, 0.50, 0.75, 1, 2, 4, 8, and 24 hours after administration of GAA and the enzyme activity was determined. AT2220 and AT2221 increased the circulating half-life of rhGAA by at least ˜2-fold. 
         FIG. 25  Western blot of recombinant GAA in plasma at 2, 8, and 24 hours after i.v. administration of GAA. 
         FIG. 26  Coadministration of DNJ or NB-DNJ with rhGAA has a similar effect on glycogen reduction. Twelve week old GAA KO mice were administered recombinant human GAA (Myozyme) 20 mg/kg i.v. every other week for 8 weeks. An oral dose of AT2220 or AT2221 (30 mg/kg) was administered 30 minutes prior to rhGAA Myozyme. Tissues were collected 21 days after the last dose of rhGAA and the level of glycogen (GAA substrate) was measured. n=5-mice/group; *p&lt;0.05 vs. untreated t-test; #p&lt;0.05 vs. Myozyme alone t-test; Dotted line indicates wild type glycogen levels. Cmax ˜40 μM following administration of 30 mg/kg AT2220 or AT2221; equivalent to approximately 600 mg in humans. 
     
    
    
     DETAILED DESCRIPTION 
     The present application provides a dosing regimen and administration schedule for the use of 1-deoxynojirimycin and enzyme replacement therapy for the treatment of Pompe disease. 
     DEFINITIONS 
     “Pompe disease,” also referred to as acid maltase deficiency, glycogen storage disease type II (GSDII), and glycogenosis type II, is a genetic lysosomal storage disorder characterized by mutations in the GAA gene which metabolizes glycogen. As used herein, this term includes infantile-, juvenile- and adult-onset types of the disease. 
     “Acid α-glucosidase” (GAA) is a lysosomal enzyme which hydrolyzes α-1,4- and α-1,6-linked-D-glucose polymers present in glycogen, maltose, and isomaltose. Alternative names are as follows: glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase; and exo-1,4-α-glucosidase, and gamma-amylase. 
     The term “rhGAA” refers to human recombinant acid α-glucosidase. Non-limiting examples of rhGAA include alglucosidase alfa and those described in U.S. Pat. No. 7,560,424 and U.S. Pat. No. 7,396,811 to Lebowitz et al., U.S. Published Application Nos. 2009/0203575, 2009/0029467, 2008/0299640, 2008/0241118, 2006/0121018, and 2005/0244400 to Lebowitz et al., U.S. Pat. Nos. 7,423,135, 6,534,300, and 6,537,785; International Published Application No. 2005/077093 and U.S. Published Application Nos. 2007/0280925, and 2004/0029779. These references are hereby incorporated by reference in their entirety 
     The term “AUC” represents a mathematical calculation to evaluate the body&#39;s total exposure over time to a given drug. In a graph plotting how concentration in the blood after dosing, the drug concentration variable lies on the y-axis and time lies on the x-axis. The area between a drug concentration curve and the x-axis for a designated time interval is the AUC. AUCs are used as a guide for dosing schedules and to compare different drugs&#39; availability in the body. 
     The term “C max ” represents the maximum plasma concentration achieved after dosing. 
     The terms “therapeutically effective dose” and “effective amount” refer to the amount of the specific pharmaceutical compound or composition that is sufficient to result in a beneficial therapeutic response. A beneficial therapeutic response can be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy, including the foregoing symptoms and surrogate clinical markers. Thus, a therapeutic response will generally be an amelioration of one or more symptoms of a disease or disorder, e.g., Pompe disease, such as those known in the art for the disease or disorder, e.g., for Pompe disease. 
     Non-limiting examples of symptoms or surrogate clinical markers of Pompe disease include: decreased GAA tissue activity; cardiomyopathy; cardiomegaly; progressive muscle weakness, especially in the trunk or lower limbs; profound hypotonia; macroglossia (and in some cases, protrusion of the tongue); difficulty swallowing, sucking, and/or feeding; respiratory insufficiency; hepatomegaly (moderate); laxity of facial muscles; areflexia; exercise intolerance; exertional dyspnea; orthopnea; sleep apnea; morning headaches; somnolence; lordosis and/or scoliosis; decreased deep tendon reflexes; lower back pain; and failure to meet developmental motor milestones. 
     The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington&#39;s Pharmaceutical Sciences” by E. W. Martin, 18th Edition, or other editions, which is hereby incorporated by reference in its entirety. 
     “1-deoxynojirimycin” (DNJ) refers to (2R,3R,4R,5S)-2-hydroxymethyl-piperidine-3,4,5-triol. As used herein, reference to “1-deoxynojirimycin” or “DNJ” throughout includes both the free base and any pharmaceutically acceptable salt forms of the same. The hydrochloride salt of DNJ is known as “1-deoxynojirimycin hydrochloride” or “DNJ HCl.” 
     “1-deoxynojirimycin derivative” or “1-DNJ derivative” or “DNJ derivative” refers to a compound with the following structure: 
     
       
         
         
             
             
         
       
     
     where R 1  is H or a straight or branched alkyl, cycloalkyl, alkenyl, alkoxyalkyl or aminoalkyl containing 1-12 carbon atoms, an aryl, alkylaryl, heteroaryl, or heteroaryl alkyl containing 5-12 ring atoms, where R 1  is optionally substituted with one or more —OH, —COOH, —Cl, —F, —CF 3 , —OCF 3 , —O—C(═O)N-(alkyl) 2 ;
 
R 2  is H; a straight or branched alkyl, cycloalkyl, alkenyl, alkylaryl, or alkoxyalkyl, containing 1-9 carbon atoms or aryl containing 5-12 carbon atoms, wherein R 2  is optionally substituted with —OH, —COOH, —CF 3 , —OCF 3  or a heterocyclic ring; and at least one of R 1  and R 2  is not H; or a pharmaceutically acceptable salt thereof.
 
     In some embodiments “1-deoxynojirimycin derivative” or “1-DNJ derivative” or “DNJ derivative” refers to is (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol or miglustat or N-butyl-DNJ. 
     The term “adjuvant” or “adjuvant therapy” refers to any additional substance, treatment, or procedure used for increasing the efficacy, safety, or otherwise facilitating or enhancing the performance of a primary substance, treatment, or procedure. 
     The term “combination therapy” refers to any therapy wherein the results are enhanced as compared to the effect of each therapy when it is performed individually. The individual therapies in a combination therapy may be administered concurrently or consecutively. 
     Enhancement may include any improvement of the effect of the various therapies that may result in an advantageous result as compared to the results achieved by the therapies when performed alone. Enhanced effect and determination of enhanced effect may be measured by various parameters such as, but not limited to: temporal parameters (e.g., length of treatment, recovery time, long-term effect of the treatment or reversibility of treatment); biological parameters (e.g., cell number, cell volume, cell composition, tissue volume, tissue size, tissue composition); spatial parameters (e.g., tissue strength, tissue size or tissue accessibility) and physiological parameters (e.g., body contouring, pain, discomfort, recovery time or visible marks). Enhanced effect may include a synergistic enhancement, wherein the enhanced effect is more than the additive effects of each therapy when performed by itself. Enhanced effect may include an additive enhancement, wherein the enhanced effect is substantially equal to the additive effect of each therapy when performed by itself. Enhanced effect may include less than a synergistic effect, wherein the enhanced effect is lower than the additive effect of each therapy when performed by itself, but still better than the effect of each therapy when performed by itself. 
     The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” can mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise. 
     Formulation and Administration 
     1-deoxynojirimycin can be administered as the free base or as a pharmacologically acceptable salt form, including 1-deoxynojirimycin hydrochloride. It can be administered in a form suitable for any route of administration, including e.g., orally in the form tablets, capsules, or liquid, or in sterile aqueous solution for injection. It can be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouth washes, or a dry powder for constitution with water or other suitable vehicle before use, optionally with flavoring and coloring agents for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications. Solid compositions such as tablets, capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules, bullets, or premix preparations can also be used. Solid and liquid compositions for oral use can be prepared according to methods well known in the art. Such compositions can also contain one or more pharmaceutically acceptable carriers and excipients which can be in solid or liquid form. When the compound is formulated for oral administration, the tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. 
     The pharmaceutically acceptable excipients also include, but are not limited to, microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrolidone, hydroxypropyl ethylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc can be included. 
     In a specific embodiment, 1-deoxynojirimycin hydrochloride is formulated with magnesium stearate and pregelatinized starch in a white, hard gelatin capsule. 
     Enzyme Replacement Therapy 
     The current approved treatment for Pompe disease is enzyme replacement therapy. Two alglucosidase alfa products are currently available for the treatment of Pompe disease: Myozyme® (Genzyme Corporation) and Lumizyme® (Genzyme Corporation). These two forms of ERT are intended to compensate for a patient&#39;s inadequate GAA activity with a recombinant form of the enzyme, administered intravenously. While ERT is effective in many settings, the rhGAA has a short circulating half-life, low tissue uptake, and can elicit immune responses that adversely affect tolerability and efficacy 
     The recommended dosage of alglucosidase alfa is 20 mg/kg body weight infused every 2 weeks as an intravenous infusion. This infusion is administered over a 4-hour period. 
     1-deoxynojirimycin hydrochloride stabilizes rhGAA in vitro and in vivo. Binding of 1-deoxynojirimycin hydrochloride to rhGAA results in significant concentration-dependent increases in the physical stability of the enzyme in a neutral pH buffer, as measured by thermal denaturation and by enzymatic activity. In addition, the stability of rhGAA in human blood is significantly increased when incubated with 1-deoxynojirimycin hydrochloride, as measured by activity (half-life is approximately 6 hours in the absence of 1-deoxynojirimycin hydrochloride, whereas in the presence of 1-deoxynojirimycin hydrochloride there was no measurable loss in enzymatic activity over a 24 hour period). 
     In rats, bolus intravenous administration of 10 mg/kg rhGAA 30 minutes after a single oral administration of 3 or 30 mg/kg 1-deoxynojirimycin hydrochloride resulted in 1.5- and 2.1-fold increases in the circulating half-life of rhGAA, respectively, as measured by activity and Western blotting. Similar effects were seen on the circulating half-life of rhGAA when 1-deoxynojirimycin hydrochloride (3 or 30 mg/kg PO) was administered to rats, followed 30 minutes later by a 1-hour intravenous infusion of rhGAA (10 mg/kg). Importantly, these doses of 1-deoxynojirimycin hydrochloride result in plasma exposure levels in the rat that are comparable to those that can be achieved following oral administration of 50 or 600 mg 1-deoxynojirimycin hydrochloride, respectively, to humans. 
     In GAA knock-out mice, oral administration of 10, 100, or 1000 mg/kg 1-deoxynojirimycin hydrochloride 30 minutes before and 8, 16, and 24 hours after bolus intravenous administration of rhGAA (10 mg/kg, once per week for up to 1, 2 or 3 weeks) resulted in significant and dose-dependent increases in tissue GAA levels as measured by activity and Western blotting 2, 4, and 7 days post-administration. Co-administration of 10 mg/kg 1-deoxynojirimycin hydrochloride (which yields exposure in mice that is comparable to that seen in humans following administration of about 200 mg) resulted in maximal GAA increases that were up to 2.5-, 2.3-, 2.2-, 4.0-, and 1.7-fold greater in heart, diaphragm, quadriceps, gastrocnemius, and triceps, respectively, compared to those seen following administration of rhGAA alone. Similar results were seen in wild-type C57BL/6 mice following a single administration of 3, 30, or 100 mg/kg 1-deoxynojirimycin hydrochloride 30 minutes prior to a single bolus intravenous administration of rhGAA (10 mg/kg). Importantly, the 3, 10, and 30 mg/kg doses of 1-deoxynojirimycin hydrochloride result in plasma exposure levels in the mouse that are comparable to those that can be achieved following oral administration of 50, 150 or 600 mg 1-deoxynojirimycin hydrochloride, respectively, to humans. 
     The effect of a single oral administration of 1-deoxynojirimycin hydrochloride on rhGAA tissue uptake was studied in GAA knock-out mice. Bolus intravenous administration of rhGAA (20 mg/kg) 30 minutes after a single oral administration of 1-deoxynojirimycin hydrochloride (30 mg/kg every other week for 8 weeks; four administrations total) resulted in a significant increase in GAA activity measured 7 days following the last injection. GAA activity increases were approximately 2.1-, 2.0-, 1.5-, 1.7-, 1.6-, and 2.0-fold greater following 1-deoxynojirimycin hydrochloride co-administration compared to administration of rhGAA alone, in heart, diaphragm, gastrocnemius, quadriceps, triceps and tongue, respectively. These data indicate that in GAA knock-out mice, administration of 30 mg/kg 1-deoxynojirimycin hydrochloride prior to administration of rhGAA every other week for 8 weeks yields significantly greater rhGAA tissue uptake as compared to those seen following rhGAA administration alone. 
     Repeat rhGAA administration studies were conducted in GAA knock-out mice to assess effects on tissue glycogen levels. Bolus intravenous administration of rhGAA (20 mg/kg) 30 minutes after a single oral administration of 1-deoxynojirimycin hydrochloride (10 or 30 mg/kg once every other week for 8 weeks resulted in a dose-dependent reduction in tissue glycogen levels measured 21 days following the last injection. Glycogen reduction was approximately 2.3-, 1.6-, 2.6-, 2.7-, 2.2-, 1.2-, 1.4-, and 1.3-fold greater in heart, diaphragm, quadriceps, gastrocnemius, triceps, soleus, biceps, and tongue, respectively, following 1-deoxynojirimycin hydrochloride co-administration compared to rhGAA alone, respectively. Similar effects were seen on tissue glycogen levels following four biweekly oral administrations of 1-deoxynojirimycin hydrochloride (30 mg/kg) followed 30 minutes later by bolus intravenous administration of rhGAA (40 mg/kg). 
     EXAMPLES 
     Example 1 
     Dosing Regimen for the Treatment of Pompe Disease Using 1-Deoxynojirimycin Hydrochloride and Alglucosidase Alfa 
     One objective of the study is to evaluate the safety, effectiveness, and pharmacodynamics of dose regimens comprising co-administering 1-deoxynojirimycin hydrochloride (also referred to as AT2220) and alglucosidase alfa in patients with Pompe disease. 
     Another objective of the study is to assess the effects of various doses of 1-deoxynojirimycin hydrochloride on the GAA activity. This will be evaluated by measuring the GAA activity in muscle after dosing with alglucosidase alfa alone and alglucosidase alfa in combination with 1-deoxynojirimycin hydrochloride, at 3 and/or 7 days after dosing, by measuring GAA activity and protein levels. 
     WBC (PBMC) GAA activity and protein levels will be measured, as well as anti-rhGAA antibody titer before initiation of an infusion of alglucosidase alfa. All plasma, WBC and muscle measurements of GAA enzyme activity are performed with and without Con A capture and determination of protein levels is by Western blot. 
     Study Design. 
     This is a Phase 2 clinical, single-dose, open-label study to assess the safety and effectiveness of co-administering 1-deoxynojirimycin hydrochloride and alglucosidase alfa. The study will be conducted in male and female subjects between 18 and 65 years of age who have been receiving a stable dose of alglucosidase alfa for at least one month before study entry. Approximately 16 subjects will be enrolled. 
     This study will be to evaluate the safety and effect of ascending doses (50, 100, 250 and 600 mg) of 1-deoxynojirimycin hydrochloride administered 1 hour before initiation of an infusion of alglucosidase alfa on the pharmacokinetics of GAA. Four subjects will be enrolled at each of the four 1-deoxynojirimycin hydrochloride dose levels. Each cohort of four subjects will receive a single intravenous infusion of alglucosidase alfa alone, followed two to four weeks later by a single oral dose of 1-deoxynojirimycin hydrochloride administered 1 hour before initiation of an intravenous infusion of alglucosidase alfa. 
     Dose escalation to the next dose level of 1-deoxynojirimycin hydrochloride may proceed following review of safety and tolerability data from the previous dose level group(s). Safety data reviewed will include adverse events (including infusion reactions), clinical laboratory tests (including creatine kinase, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4, 12-Lead ECGs, physical examinations, muscle strength tests and vital signs. 
     Each subject will receive alglucosidase alfa alone as an intravenous infusion in Period 1 and a single dose of 1-deoxynojirimycin hydrochloride one hour before initiation of an intravenous infusion of alglucosidase alfa in Period 2. The dose of alglucosidase alfa administered in Periods 1 and 2 will be identical. 
     Each cohort will consist of 4 subjects. Subjects will be enrolled sequentially to one of the four dosing cohorts to 1-deoxynojirimycin hydrochloride at the following dose levels in Period 2: 
     Cohort 1: A single 50 mg oral dose of 1-deoxynojirimycin hydrochloride; 
     Cohort 2: A single 100 mg oral dose of 1-deoxynojirimycin hydrochloride; 
     Cohort 3: A single 250 mg oral dose of 1-deoxynojirimycin hydrochloride; 
     Cohort 4: A single 600 mg oral dose of 1-deoxynojirimycin hydrochloride. 
     For Period 1, prior to their next scheduled alglucosidase alfa infusion, subjects will have the following assessments performed: adverse event assessment, concomitant medications, physical exam, weight, vital signs, 12-lead ECG, clinical laboratory tests (including creatine analysis, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4 and muscle strength tests. 
     On the morning of Day 1, the subject&#39;s current alglucosidase alfa dose will be administered as an infusion using an infusion pump. The infusion rate (and any changes in rate during the infusion), infusion duration and dose of alglucosidase alfa administered should be identical in Periods 1 and 2. Blood samples for pharmacokinetic analysis will be collected immediately before initiation of the alglucosidase alfa infusion and over a 24-hour period after initiation of the alglucosidase alfa infusion. Plasma and WBC GAA enzyme activity and plasma anti-rhGAA antibody titer will be determined from the collected blood samples at the times summarized in Table 2. A 12-lead ECG will be performed at the end of the alglucosidase alfa infusion, immediately after collection of the post-infusion blood sample. 
     After collection of the last pharmacokinetic sample, the following assessments will be performed: adverse event assessment (including infusion reactions), concomitant medications, vital signs, 12-lead ECG, clinical laboratory tests (including creatine kinase, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4 and muscle strength tests. 
     On Day 7, subjects will have the following assessments performed: adverse event assessment, concomitant medications, physical exam, vital signs, clinical laboratory tests (including creatine kinase, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4 and muscle strength tests. A muscle biopsy will be collected from which GAA enzyme activity will be determined. A blood sample for plasma and WBC GAA enzyme level determinations will also be collected. 
     A muscle biopsy will also be collected from which GAA enzyme activity and 1-deoxynojirimycin hydrochloride levels will be determined, on Day 3. 
     For Period 2, prior to their next scheduled alglucosidase alfa infusion, approximately 2 weeks after the administration of the alglucosidase alfa infusion in Period 1, subjects will have the following assessments performed: adverse event assessment, concomitant medications, physical exam, weight, vital signs, 12-lead ECG, clinical laboratory tests (including creatine analysis, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4 and muscle strength tests. 
     On the morning of Day 1, an oral dose of 1-deoxynojirimycin hydrochloride will be administered 1 hour prior to the scheduled alglucosidase alfa infusion. Subjects will fast for at least 2 hours before and 2 hours after 1-deoxynojirimycin hydrochloride administration. The infusion rate (and any changes in rate during the infusion), infusion duration and dose of alglucosidase alfa administered should be identical in Periods 1 and 2. 
     Blood samples for pharmacokinetic analysis will be collected before dosing 1-deoxynojirimycin hydrochloride and at 1 hour after administration of 1-deoxynojirimycin hydrochloride (i.e., immediately before initiation of the alglucosidase alfa infusion) and over the 24-hour period after initiation of the alglucosidase alfa infusion. Plasma and WBC GAA enzyme activity, plasma 1-deoxynojirimycin hydrochloride concentrations and plasma anti-rhGAA antibody titer will be determined from the collected blood samples at the times summarized in Table 2. A 12-lead ECG will be performed at the end of the alglucosidase alfa infusion, immediately after collection of the post-infusion blood sample. 
     After collection of the last pharmacokinetic sample, the following assessments will be performed: adverse event assessment (including infusion reactions), concomitant medications, vital signs, 12-lead ECG, clinical laboratory tests (including creatine kinase, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4 and muscle strength tests. 
     On Day 7, subjects will have the following assessments performed: adverse event assessment, concomitant medications, physical exam, vital signs, clinical laboratory tests (including creatine kinase, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4 and muscle strength tests. A muscle biopsy will be collected from which GAA enzyme activity and 1-deoxynojirimycin hydrochloride levels will be determined. A blood sample for plasma and WBC GAA enzyme level determinations will also be collected. 
     A muscle biopsy will also be collected from which GAA enzyme activity and 1-deoxynojirimycin hydrochloride levels will be determined, on Day 3. 
     In the follow-up 26 to 30 days after dosing with 1-deoxynojirimycin hydrochloride and alglucosidase alfa in Period 2, subjects will have the following assessments performed: adverse event assessment, concomitant medications, physical exam, vital signs, 12-lead ECG, clinical laboratory tests (including creatine kinase, LDH (LDH-5), alkaline phosphatase, AST, ALT), Hex4, muscle strength tests and anti-rhGAA antibody titers. 
     Assessment and Sample Collection Schedules. 
     Table 1 shows the schedule of assessments for Periods 1 and 2. Sample collection times and analytes for co-administration of 1-deoxynojirimycin hydrochloride with alglucosidase alfa are shown in Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Schedule of Assessments 
               
            
           
           
               
               
               
               
            
               
                   
                 Screening 
                   
                 Follow-up 
               
               
                   
                 Within 28 
                   
                 26-30 days 
               
               
                   
                 days of 
                 Periods 1 and 2 
                 after dosing 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Activity 
                 Enrollment 
                 Day −1 
                 Day 1 
                 Day 2 
                 Day 7 
                 in Period 2 
               
               
                   
               
               
                 Informed Consent 
                 X 
                   
                   
                   
                   
                   
               
               
                 Medical History 
                 X 
               
               
                 and Demographic 
               
               
                 Data 
               
               
                 Physical Exam 
                 X 
                 X 
                   
                   
                 X 
                 X 
               
               
                 ECG (12-lead) 
                 X 
                 X 
                 X 
                 X 
                   
                 X 
               
               
                 Vital Signs 1   
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Hematology 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
               
               
                 Urinalysis 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
               
               
                 Serum Chemistry 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
               
               
                 Hex4 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
               
               
                 Record of 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Concomitant 
               
               
                 Medication 
               
               
                 eGFR 
                 X 
               
               
                 Drug Dose 
                   
                   
                     X 2   
               
               
                 PK Blood 
                   
                   
                 X 
                 X 
                 X 
               
               
                 Sampling 3   
               
               
                 Muscle Strength 
                 X 
                 X 
                   
                 X 
                 X 
                 X 
               
               
                 Test 
               
               
                 (dynamometer) 
               
               
                 Muscle Biopsy 
                   
                   
                   
                   
                 X 
               
               
                 Antibody Titer 
                   
                   
                 X 
                   
                   
                 X 
               
               
                 Adverse Events 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
               
               
                   1 Vital signs include temperature, blood pressure, heart rate and respiration. 
               
               
                   2 Drug(s) administered: alglucosidase alfa (Periods 1 and 2), 1-deoxynojirimycin hydrochloride (Period 2). 
               
               
                   3 Sample collection times and analytes are summarized in Table 2. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Blood and Muscle Collection Times and Sample Analysis 
               
               
                   
               
             
            
               
                 Period 1 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Post-Initiation of Alglucosidase 
                   
               
               
                   
                   
                   
                 alfa Infusion (hr) 
                 Day 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Sample Collected 
                 Predose 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 12 
                 24 
                 7 
               
               
                   
               
               
                 Blood 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Muscle 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Alglucosidase 
                   
                 &lt;------------------&gt; 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 alfa infusion 1   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Plasma 
                 Plasma GAA 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 WBC GAA 
                 X 
                   
                   
                   
                 X 
                   
                   
                   
                   
                   
                   
                   
                 X 
                 X 
               
               
                   
                 Antibody 
                 X 
               
               
                 Muscle 
                 GAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
               
                   
               
            
           
           
               
            
               
                 Period 2 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Predose 
                   
                 Post-Initiation of Alglucosidase 
                   
               
               
                   
                   
                 DNJ 
                   
                 alfa Infusion (hr) 
                 Day 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Sample Collected 
                 HCl 
                 0 2   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 12 
                 24 
                 7 
               
               
                   
               
               
                 Blood 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Muscle 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Alglucosidase 
                   
                   
                 &lt;---------------&gt; 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 alfa infusion 1,3   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 DNJ HCl Dose 
                   
                 X 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Plasma 
                 Plasma GAA 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 WBC GAA 
                 X 
                 X 
                   
                   
                   
                 X 
                   
                   
                   
                   
                   
                   
                   
                 X 
                 X 
               
               
                   
                 Antibody 
                 X 
               
               
                   
                 DNJ HCl 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 Muscle 
                 GAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
               
                   
                 DNJ HCl 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 X 
               
               
                   
               
               
                   1 Duration of infusion for individual subjects may differ from 4 hours, however, infusion duration and infusion rate should be identical in both periods. An additional blood sample immediate before termination of the infusion (to be assayed for plasma GAA only) will be collected if the duration of infusion varies from 4 hours and the end of infusion does not coincide with a pre-established blood sample. 
               
               
                   2 Sample collected 1 hour after 1-deoxynojirimycin hydrochloride dose, immediately before initiation of alglucosidase alfa infusion. 
               
               
                   3 Alglucosidase alfa infusion started one hour after administration of 1-deoxynojirimycin hydrochloride. 
               
            
           
         
       
     
     1-Deoxynojirimycin Hydrochloride and GAA Plasma Pharmacokinetics. 
     Concentrations of 1-deoxynojirimycin hydrochloride in blood samples will be measured in plasma using a validated LC-MS/MS assay. GAA activity in plasma will be determined by a validated assay measuring enzyme activity using 4-MUG, with and without Con A. GAA protein levels will be measured by Western blotting using anti-human GAA antibody. 
     GAA Enzyme Activity and 1-Deoxynojirimycin Hydrochloride Levels in Muscle. 
     GAA enzyme activity will be examined in muscle biopsy samples. One piece of muscle tissue will be removed as described in Table 1. GAA activity in muscle will be determined by a validated assay measuring enzyme activity using 4-MUG, with and without Con A. GAA protein levels will be measured by Western blotting using anti-human GAA antibody. 1-deoxynojirimycin hydrochloride concentrations in muscle samples collected in Period 2 will be determined using a validated LC-MS/MS assay. 
     WBC (PBMC)GAA Activity. 
     GAA activity in WBCs will be determined in blood samples by a validated assay measuring enzyme activity using 4-MUG, with and without Con A. GAA protein levels will be measured by Western blotting using anti-human GAA antibody. 
     Ant-rhGAA Antibody Titer. 
     Blood samples will be collected and anti-rhGAA antibody titer will be measured in the samples described in Table 2. 
     Safety Parameters. 
     Safety parameters will be assessed by review of changes in physical exam findings, vital signs, ECG changes over time, clinical labs, Hex4 and adverse events. 
     Vital Signs, Weight and Height. 
     Body temperature and respirations will be measured at screening and check-in. To monitor safety, body temperature, respiration, seated blood pressure and heart rate will be measured before dosing and approximately 1, 2, 3, 4, and 6 hours following administration of alglucosidase alfa (Period 1) or 1-deoxynojirimycin hydrochloride (Periods 2), and on the days described in Table 1. Where the time of vital sign monitoring coincides with a blood draw, the blood draw takes precedence and the vital signs will be adjusted accordingly. 
     ECG Monitoring. 
     ECG monitoring will be performed with a standard 12-lead ECG. 
     Clinical Laboratory Tests. 
     Blood samples for clinical laboratory tests (hematology, serum chemistry) and urinalyses will be collected according to the schedule in Table 1.
         Hematology tests include total hemoglobin, hematocrit, erythrocyte, platelet and leukocyte counts with differential.   Coagulation (screening only) includes INR and aPTT.   Serum chemistry includes measurement of AST, ALT, alkaline phosphatase, total bilirubin, creatinine, creatine kinase, urea, glucose, calcium, sodium, potassium, magnesium, total protein, albumin, bicarbonate, LDH (LDH-5), blood urea nitrogen, chloride, and phosphate.   Urinalysis includes color, appearance, specific gravity, pH, protein, glucose, ketones, blood, leukocyte esterase, nitrite, bilirubin, urobilinogen and microscopy of sediment       

     Urinary Tetrasaccharides (Hex4). 
     Urine samples for Hex4 determinations will be collected at the times indicated in Table 1. 
     Muscle Strength Tests (Handheld Dynamometer). 
     Muscular strength tests, assessed by hand held dynamometer, will be conducted at Screening, Days −1 and 7 of each period and at Follow-up. Proximal and distal muscle groups will be evaluated. 
     Pharmacokinetic Parameters. 
     Non-compartmental pharmacokinetic parameters of AUC 0-t , AUC infinity , C max , t max , k el  and half-life will be calculated from plasma 1-deoxynojirimycin hydrochloride concentrations and plasma rhGAA enzyme levels. Pharmacokinetic parameters will be summarized by treatment using descriptive statistics. The AUC 0-t , AUC infinity  ratios for GAA enzyme activity alone or after administration with 1-deoxynojirimycin hydrochloride will be calculated. Pharmacokinetic and pharmacodynamic data for those subjects receiving Myozyme® and Lumizyme® will be analyzed separately. 
     Statistical Analysis. 
     The descriptive statistics (N, mean, standard deviation, and coefficient of variation, standard error, median, minimum and maximum) will be provided as appropriate. The effect of 1-deoxynojirimycin hydrochloride on GAA enzyme activity will be evaluated by calculation of the individual (by subject) AUC and C max  ratios as follows: 
     
       
         
           
             
               AUC 
                
               
                   
               
                
               Ratio 
             
             = 
             
               
                 AUC 
                 
                   infinity 
                    
                   
                       
                   
                    
                   
                     ( 
                     combination 
                     ) 
                   
                 
               
               
                 AUC 
                 
                   infinity 
                    
                   
                     ( 
                     alone 
                     ) 
                   
                 
               
             
           
         
       
       
         
           
             
               C 
                
               
                   
               
                
               max 
                
               
                   
               
                
               Ratio 
             
             = 
             
               
                 C 
                 
                   max 
                    
                   
                       
                   
                    
                   
                     ( 
                     combination 
                     ) 
                   
                 
               
               
                 C 
                 
                   max 
                    
                   
                     ( 
                     alone 
                     ) 
                   
                 
               
             
           
         
       
     
     The AUC and C max  ratios will be expressed as a mean of the individual ratios and 90% confidence interval for the mean. Pharmacokinetic and pharmacodynamic data for those subjects receiving Myozyme® and Lumizyme® will be analyzed separately. GAA activity in muscle with and without co-administration of 1-deoxynojirimycin hydrochloride will be compared. Results will be presented in tabular and graphic forms, as appropriate. All subjects who will be dosed with study medication and have sufficient data to generate reliable pharmacokinetic parameters will be included in the safety and pharmacokinetic analysis. 
     The present application is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the application in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. 
     It is further to be understood that all values are approximate, and are provided for description. 
     Example 2 
     Dosing Regimen for the Treatment of Pompe Disease Using 1-Deoxynojirimycin Hydrochloride and Alglucosidase Alfa—Cohorts 1 and 2 
     One objective of the study was to evaluate the safety, effectiveness, and pharmacodynamics of dose regimens comprising co-administering 1-deoxynojirimycin hydrochloride and alglucosidase alfa in patients with Pompe disease. 
     Another objective of the study was to assess the effects of various doses of 1-deoxynojirimycin hydrochloride on the GAA activity. This was evaluated by measuring the GAA enzyme activity and protein levels in skeletal muscle at Day 3 and/or Day 7 following a single intravenous infusion with alglucosidase alfa alone and after pre-administration of single ascending oral doses of 1-deoxynojirimycin hydrochloride. 
     Methods. 
     The study was conducted essentially according to the methods described in Example 1. Cohort 1 comprised 4 subjects. Cohort 2 comprised 6 subjects. Each subject received alglucosidase alfa alone as an intravenous infusion in Period 1 and a single 50 mg dose (Cohort 1) or 100 mg (Cohort 2) of 1-deoxynojirimycin hydrochloride one hour before initiation of an intravenous infusion of alglucosidase alfa in Period 2. The genotype, including nucleotide and amino acid changes, for each subject (where available) is shown below: 
     GAA Genotypes 
       
     
       
         
           
               
               
               
             
               
                   
               
               
                 Patient ID 
                 Nucleotide Change 
                 Amino Acid Change 
               
               
                   
               
             
            
               
                 A 
                 c.−32−13T&gt;G 
                 Splicing Mutation 
               
               
                 B 
                 Not Available 
                 — 
               
               
                 C 
                 Not Available 
                 — 
               
               
                 D 
                 c.1222A&gt;G/c.−32−13T&gt;G 
                 M408V/Splicing Mutation 
               
               
                 E 
                 c.−32−13T&gt;G 
                 Splicing Mutation 
               
               
                 F 
                 Not Available 
                 — 
               
               
                 G 
                 c.−32−13T&gt;G/c.1445C&gt;G 
                 Splicing Mutation/P482R 
               
               
                 H 
                 Not Available 
                 — 
               
               
                 I 
                 c.692+5G&gt;T/c.1211A&gt;G 
                 Splicing Mutation/D404G 
               
               
                 J 
                 Not Available 
                 — 
               
               
                   
               
            
           
         
       
     
     Results: Cohort 1 
     Plasma rhGAA Activity Increases with Co-Administration of 1-Deoxynojirimycin Hydrochloride and Acid α-Glucosidase Relative to Acid α-Glucosidase Alone 
     For co-administration of acid α-glucosidase with 50 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), a 1.5-fold mean increase in plasma rhGAA activity AUC (Area Under the Curve) was observed, as shown in Table 3. Table 3 also shows the fold change in rhGAA activity between Periods 1 and 2 in Day 7 muscle biopsies of Cohort 1 subjects. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Mean Plasma rhGAA Activity PK Summary for Cohort 1 (4 subjects): 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 C max   a   
                   
                 AUC 0-T   a   
                 AUC 0-∞   a   
                   
                   
               
               
                   
                 (nmol/ 
                 T max   b   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 T½ c   
               
               
                 Treatment 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   d   
               
               
                   
               
               
                 20 mg/kg GAA Alone 
                 16441 (31.6) 
                 5.0 (4-5) 
                 105834 (30.7) 
                 107707 (31.1) 
                 3.9 (0.6) 
                 — 
               
               
                 20 mg/kg GAA + 
                 19922 (24.4) 
                 5.0 (4-6) 
                 152207 (21.3) 
                 157162 (22.4) 
                 4.2 (0.8) 
                 1.5 
               
               
                 50 mg AT2220 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Muscle GAA 
                   
               
               
                   
                   
                   
                 Biopsy 
                 Activity 
                 Fold 
               
               
                   
                 Treatment 
                 Period 
                 Day 
                 (pmol/mg/hr) 
                 change 
               
               
                   
                   
               
               
                   
                 rhGAA Alone 
                 1 
                 7 
                 1243 (302) 
               
               
                   
                 rhGAA + 
                 2 
                 7 
                 1211 (216) 
                 1.02 (0.30) 
               
               
                   
                 50 mg AT2220 
               
               
                   
                   
               
               
                   
                   a Geometric mean (CV %) 
               
               
                   
                   b Median (range) 
               
               
                   
                   c Arithmetic mean (SD) 
               
               
                   
                   d F rel  = Geometric mean of the AUC 0-∞  ratios 
               
            
           
         
       
     
     For co-administration of acid α-glucosidase with 50 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), individual subject increases in plasma rhGAA activity AUC were 1.2-, 1.5-, 1.5-, and 1.6-fold, as shown in Tables 4-7. Tables 4-7 also show the fold change in rhGAA activity between Periods 1 and 2 in Day 7 muscle biopsies. 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2031-0101 (Patient A) (Cohort 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 T½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 20.2 mg/kg GAA Alone 
                 17415 
                 5.0 
                 109403 
                 111239 
                 3.9 
               
               
                 (1450, 4:59, 112) 
               
               
                 20.2 mg/kg GAA + 
                 20725 
                 5.0 
                 165304 
                 170617 
                 4.3 
                 1.5 
               
               
                 50 mg AT2220 
               
               
                 (1450, 5:03, 112) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Muscle GAA 
                   
               
               
                   
                   
                 Biopsy 
                 Activity 
                 Fold 
               
               
                 Treatment 
                 Period 
                 Day 
                 (pmol/mg/hr) 
                 change 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 7 
                 1352.8 
               
               
                 rhGAA + 
                 2 
                 7 
                 1398.5 
                 1.03 
               
               
                 50 mg AT2220 
               
               
                   
               
               
                 Body Weight: 71.8 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2031-0102 (Patient B) (Cohort 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 T½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 19.5 mg/kg GAA Alone 
                 19139 
                 5.0 
                 125684 
                 126956 
                 3.3 
               
               
                 (1750, 4:58, 112) 
               
               
                 19.5 mg/kg GAA + 
                 19321 
                 6.0 
                 179229 
                 190630 
                 5.3 
                 1.5 
               
               
                 50 mg AT2220 
               
               
                 (1750, 5:10, 112) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Muscle GAA 
                   
               
               
                   
                   
                 Biopsy 
                 Activity 
                 Fold 
               
               
                 Treatment 
                 Period 
                 Day 
                 (pmol/mg/hr) 
                 change 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 7 
                 1010.5 
               
               
                 rhGAA + 
                 2 
                 7 
                 956.3 
                 0.95 
               
               
                 50 mg AT2220 
               
               
                   
               
               
                 Body weight: 89.8 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2031-0103 (Patient C) (Cohort 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 T½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 20.6 mg/kg GAA Alone 
                 10495 
                 5.0 
                 68617 
                 69526 
                 3.7 
               
               
                 (1150, 5:00, 112) 
               
               
                 20.6 mg/kg GAA + 
                 14808 
                 5.0 
                 111781 
                 114447 
                 4 
                 1.6 
               
               
                 50 mg AT2220 
               
               
                 (1150, 5:04, 112) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Muscle GAA 
                   
               
               
                   
                   
                 Biopsy 
                 Activity 
                 Fold 
               
               
                 Treatment 
                 Period 
                 Day 
                 (pmol/mg/hr) 
                 change 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 7 
                 988.4 
               
               
                 rhGAA + 
                 2 
                 7 
                 1383.1 
                 1.40 
               
               
                 50 mg AT2220 
               
               
                   
               
               
                 Body Weight: 55.8 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 6003-0101 (Patient D) (Cohort 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 T½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 19.7 mg/kg GAA Alone 
                 20889 
                 4.0 
                 132970 
                 137064 
                 4.6 
               
               
                 (2150, 3:50, 175) 
               
               
                 19.7 mg/kg GAA + 
                 26568 
                 4.0 
                 162063 
                 163896 
                 3.4 
                 1.2 
               
               
                 50 mg AT2220 
               
               
                 (2150, 3:50, 175) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Muscle GAA 
                   
               
               
                   
                   
                 Biopsy 
                 Activity 
                 Fold 
               
               
                 Treatment 
                 Period 
                 Day 
                 (pmol/mg/hr) 
                 change 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 7 
                 1620.4 
               
               
                 rhGAA + 
                 2 
                 7 
                 1109.0 
                 0.68 
               
               
                 50 mg AT2220 
               
               
                   
               
               
                 Body weight 109.0 kg 
               
            
           
         
       
     
     Table 8 below shows the cumulative AUCs for rhGAA activity in plasma of cohort 1. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Time 
                 2031-0101 
                 2031-0102 
                 2031-0103 
                 6003-0101 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 995 
                 797 
                 1498 
                 1317 
                 1094 
                 840 
                 1528 
                 1296 
               
               
                 2 
                 5514 
                 4620 
                 7422 
                 7154 
                 4549 
                 4462 
                 8617 
                 7784 
               
               
                 3 
                 14322 
                 13802 
                 18433 
                 18370 
                 10513 
                 12120 
                 23531 
                 21747 
               
               
                 4 
                 27215 
                 28049 
                 33967 
                 33603 
                 19342 
                 23519 
                 43327 
                 43801 
               
               
                 5 
                 43537 
                 46504 
                 52486 
                 50460 
                 29815 
                 37448 
                 62700 
                 67755 
               
               
                 6 
                 58363 
                 66269 
                 68174 
                 68442 
                 39357 
                 51079 
                 76972 
                 87223 
               
               
                 7 
                 69498 
                 83355 
                 79546 
                 86144 
                 46438 
                 62230 
                 86420 
                 102711 
               
               
                 8 
                 78151 
                 97251 
                 88568 
                 100655 
                 51167 
                 71026 
                 93589 
                 114607 
               
               
                 9 
                 84748 
                 109173 
                 95606 
                 112503 
                 54568 
                 77778 
                 99912 
                 123747 
               
               
                 10 
                 89665 
                 119039 
                 101047 
                 122588 
                 57196 
                 83434 
                 105495 
                 131397 
               
               
                 12 
                 96302 
                 133401 
                 109601 
                 138339 
                 61036 
                 92644 
                 113716 
                 142712 
               
               
                 24 
                 109403 
                 165304 
                 125684 
                 179229 
                 68617 
                 111781 
                 132970 
                 162063 
               
               
                   
               
            
           
         
       
     
     Table 9 shows the Total rhGAA Protein in Plasma PK Summary by Western Blot (Cohort 1) 
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
             
            
               
                 Period 1 (Treatment = rhGAA Alone) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 C max   
                 T max   
                 AUC 0-T   
                 AUC 0-∞   
                 T½ 
               
               
                 SubjID 
                 (ng/mL) 
                 (hr) 
                 (ng*hr/mL) 
                 (ng*hr/mL) 
                 (hr) 
               
               
                   
               
               
                 2031-0101 
                 197632 
                 5 
                 1573875 
                 1842902 
                 8.9 
               
               
                 2031-0102 
                 240858 
                 5 
                 2035347 
                 2235425 
                 6.9 
               
               
                 2031-0103 
                 266201 
                 5 
                 1907938 
                 2142094 
                 2.7 
               
               
                 6003-0101 
                 507039 
                 5 
                 2513158 
                 2523836 
                 0.8 
               
               
                 N 
                 4 
                 4 
                 4 
                 4 
                 4 
               
               
                 Mean 
                 302932 
                 5.0 
                 2007580 
                 2186064 
                 4.8 
               
               
                 SD 
                 138984 
                 0.0 
                 389191 
                 280606 
                 3.7 
               
               
                 Min 
                 197632 
                 5.0 
                 1573875 
                 1842902 
                 0.8 
               
               
                 Median 
                 253529 
                 5.0 
                 1971643 
                 2188760 
                 4.8 
               
               
                 Max 
                 507039 
                 5.0 
                 2513158 
                 2523836 
                 8.9 
               
               
                 CV % 
                 45.9 
                 0.0 
                 19.4 
                 12.8 
                 77.2 
               
               
                 Geometric 
                 283118 
                 5.0 
                 1979695 
                 2172404 
                 3.4 
               
               
                 Mean 
               
               
                   
               
            
           
           
               
            
               
                 Period 2 (Treatment = rhGAA + 50 mg AT2220) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 C max   
                 T max   
                 AUC 0-T   
                 AUC 0-∞   
                   
                 T½ 
               
               
                 SubjID 
                 (ng/mL) 
                 (hr) 
                 (ng*hr/mL) 
                 (ng*hr/mL) 
                 F rel   
                 (hr) 
               
               
                   
               
               
                 2031-0101 
                 259022 
                 6.0 
                 2440258 
                 2712963 
                 1.5 
                 6.8 
               
               
                 2031-0102 
                 281085 
                 5.0 
                 3037230 
                 3344934 
                 1.5 
                 6.3 
               
               
                 2031-0103 
                 334448 
                 6.0 
                 2400468 
                 2903123 
                 1.4 
                 4 
               
               
                 6003-0101 
                 645181 
                 4.0 
                 3437632 
                 3618220 
                 1.4 
                 2 
               
               
                 N 
                 4 
                 4 
                 4 
                 4 
                 4 
                 4 
               
               
                 Mean 
                 379934 
                 5.3 
                 2828897 
                 3144810 
                 1.4 
                 4.8 
               
               
                 SD 
                 179644 
                 1.0 
                 499517 
                 411935 
                   
                 2.2 
               
               
                 Min 
                 259022 
                 4.0 
                 2400468 
                 2712963 
                   
                 2.0 
               
               
                 Median 
                 307767 
                 5.5 
                 2738744 
                 3124029 
                   
                 5.2 
               
               
                 Max 
                 645181 
                 6.0 
                 3437632 
                 3618220 
                   
                 6.8 
               
               
                 CV % 
                 47.3 
                 18.2 
                 17.7 
                 13.1 
                   
                 46.4 
               
               
                 Geometric 
                 354035 
                 5.2 
                 2796515 
                 3124625 
                   
                 4.3 
               
               
                 Mean 
               
               
                   
               
            
           
         
       
     
     Results: Cohort 2 
     Plasma rhGAA Activity Increases with Co-Administration of 1-Deoxynojirimycin Hydrochloride and Acid α-Glucosidase Relative to Acid α-Glucosidase Alone 
     For co-administration of acid α-glucosidase with 100 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), a 1.7-fold mean increase in plasma rhGAA activity AUC (Area Under the Curve) was observed, as shown in Table 10A. Table 10A also shows the fold change in rhGAA activity between Periods 1 and 2 in Day 3 and Day 7 muscle biopsies of Cohort 2 subjects. Table 10B shows the plasma PK summary for 1-deoxynojirimycin hydrochloride from Period 2 of Cohorts 1 and 2. Table 10C shows the total rhGAA protein concentrations from Cohorts 1 and 2 as measured by Western blot. 
     
       
         
           
               
             
               
                 TABLE 10A 
               
               
                   
               
               
                 Mean Plasma rhGAA Activity PK Summary for Cohorts 1 (4 subjects) and Cohort 2 
               
               
                 (6 subjects), and Mean Muscle Biopsy rhGAA Activity for Cohort 2 (6 subjects): 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   a   
                   
                 AUC 0-T   a   
                 AUC 0-∞   a   
                   
                   
               
               
                 Period/ 
                   
                 (nmol/ 
                 T max   b   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 
                 t½ c   
               
               
                 Treatment 
                 Cohort 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio a   
                 (hr) 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 16441 (31.6) 
                 5.0 (4-5) 
                 105834 (30.7) 
                 107707 (31.1) 
                 — 
                 3.9 (0.6) 
               
               
                 (N = 4) 
               
               
                 rhGAA + 
                 1 
                 19922 (24.4) 
                 5.0 (4-6) 
                 152207 (21.3) 
                 157162 (22.4) 
                 1.5 (13.2) 
                 4.2 (0.8) 
               
               
                 50 mg AT2220 
               
               
                 (N = 4) 
               
               
                 rhGAA Alone 
                 2 
                 22785 (18.0) 
                 4.0 (3-5) 
                 142415 (30.4) 
                 144953 (31.5) 
                 — 
                 3.8 (0.6) 
               
               
                 (N = 6) 
               
               
                 rhGAA + 
                 2 
                 28607 (13.9) 
                 4.0 (3-6) 
                 229434 (25.3) 
                 241203 (27.5) 
                 1.7 (10.1) 
                 4.9 (0.8) 
               
               
                 100 mg AT2220 
               
               
                 (N = 6) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
               
               
                   
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
               
               
                   
                   
               
               
                   
                 rhGAA Alone 
                 — 
                 1538.8 
               
               
                   
                 rhGAA + 
                 3 
                 2846.1 
                 1.3 
               
               
                   
                 100 mg AT2220 
               
               
                   
                 rhGAA + 
                 7 
                 1115.0 
                 1.2 
               
               
                   
                 100 mg AT2220 
               
               
                   
                 Follow-up for 
                 28 
                 634.5 
               
               
                   
                 Baseline 
               
               
                   
                   
               
               
                   
                   a Geometric mean (CV %) 
               
               
                   
                   b Median (range) 
               
               
                   
                   c Arithmetic mean (SD) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10B 
               
             
            
               
                   
               
               
                 Mean AT2220 PK Summary for Cohorts 1 and 2 
               
               
                 AT2220 PK Summary Table 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 C max   a   
                 T max   b   
                 AUC 0-T   a   
                 AUC 0-∞   a   
                 AUC 0-∞   
                 t½ c   
               
               
                 AT2220 Dose 
                 (ng/mL) 
                 (hr) 
                 (ng · hr/mL) 
                 (ng · hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
               
                  50 mg (N = 4) 
                 1035 (25.0) 
                 2.0 (1-3) 
                  6972 (28.1) 
                  7008 (28.2) 
                 — 
                 3.1 (0.2) 
               
               
                 100 mg (N = 6) 
                 1863 (13.1) 
                 3.0 (1-4) 
                 13003 (18.9) 
                 13137 (19.2) 
                 1.9 
                 3.4 (0.3) 
               
               
                   
               
               
                   a Geometric mean (CV %) 
               
               
                   b Median (range) 
               
               
                   c Arithmetic mean (SD) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10C 
               
             
            
               
                   
               
               
                 Cohorts 1 and 2 Plasma rhGAA Protein PK Summary 
               
               
                 Cohorts 1 and 2 Plasma rhGAA Protein PK Summary 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   a   
                   
                 AUC 0-T   a   
                   
                 AUC 0-∞   a   
                   
               
               
                 Period/ 
                   
                 (nmol/ 
                 T max   b   
                 (hr*nmol/ 
                 AUC 0-T   a   
                 (hr*nmol/ 
                 T½ c   
               
               
                 Treatment 
                 Cohort 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 Ratio 
                 hr/mL) 
                 (hr) 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 283118 (45.9) 
                 5.0 (5-5) 
                 1979695 (19.4) 
                 — 
                 2172404 (12.8) 
                 4.8 (3.7) 
               
               
                 (N = 4) 
               
               
                 rhGAA + 
                 1 
                 354035 (47.3) 
                 5.5 (4-6) 
                 2796515 (17.7) 
                 1.4 (9.2)  
                 3124625 (13.1) 
                 4.8 (2.2) 
               
               
                 50 mg AT2220 
               
               
                 (N = 4) 
               
               
                 rhGAA Alone 
                 2 
                 399623 (25.8) 
                 4.0 (4-5) 
                 2315249 (44.3) 
                 — 
                 2862876 (86.8) 
                 3.9 (4.5) 
               
               
                 (N = 6) 
               
               
                 rhGAA + 
                 2 
                 430738 (30.5) 
                 4.0 (4-7) 
                 3841773 (44.5) 
                 1.7 (18.5) 
                 5123518 (42.3) 
                 6.1 (3.2) 
               
               
                 100 mg AT2220 
               
               
                 (N = 6) 
               
               
                   
               
               
                   a Geometric mean (CV %) 
               
               
                   b Median (range) 
               
               
                   c Arithmetic mean (SD) 
               
            
           
         
       
     
     For co-administration with 100 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), individual subject increases in plasma rhGAA activity AUC were 1.5-, 1.5-, 1.6-, 1.7-, 1.8- and 1.9-fold, as shown in Tables 11-16. Tables 11-16 also show the fold change in rhGAA activity between Periods 1 and 2 in Day 3 or Day 7 muscle biopsies of Cohort 2 subjects. 
     
       
         
           
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2028-0201 (Patient F) (Cohort 2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 t½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 18.2 mg/kg GAA alone 
                 20541 
                 4.0 
                 125868 
                 127210 
                 3.5 
                 — 
               
               
                 (1850, 4:00, 175) 
               
               
                 18.2 mg/kg GAA + 
                 28053 
                 4.0 
                 212384 
                 222194 
                 4.8 
                 1.75 
               
               
                 100 mg AT2220 
               
               
                 (1850, 3:57, 175) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Baseline- 
                 % Change 
               
               
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
                 subtracted 
                 from 
               
               
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
                 Ratio 
                 Baseline 
               
               
                   
               
               
                 rhGAA Alone 
                 7 
                 756.4 
                   
                   
                 13.6 
               
               
                 rhGAA + 
                 7 
                 794.1 
                 1.0 
                 1.4 
                 19.2 
               
               
                 100 mg AT2220 
               
               
                 Follow-up for 
                 28 
                 666.0 
               
               
                 Baseline 
               
               
                   
               
               
                 Body weight: 101.5 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2029-0202 (Patient G) (Cohort 2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 t½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 31.3 mg/kg GAA alone 
                 29167 
                 5.0 
                 231174 
                 240183 
                 4.6 
                 — 
               
               
                 (2200, 5:12, 115) 
               
               
                 31.3 mg/kg GAA + 
                 34882 
                 6.0 
                 321065 
                 349917 
                 5.9 
                 1.46 
               
               
                 100 mg AT2220 
               
               
                 (2200, 5:24, 115) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Baseline- 
                 % Change 
               
               
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
                 subtracted 
                 from 
               
               
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
                 Ratio 
                 Baseline 
               
               
                   
               
               
                 rhGAA Alone 
                 7 
                 1019.9 
                   
                   
                 178 
               
               
                 rhGAA + 
                 7 
                 1608.9 
                 1.6 
                 1.9 
                 339 
               
               
                 100 mg AT2220 
               
               
                 Follow-up forBaseline 
                 28 
                 366.8 
               
               
                   
               
               
                 Body weight: 70.4 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 6003-0202 (Patient I) (Cohort 2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 t½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 20.0 mg/kg GAA 
                 20799 
                 4.0 
                 117942 
                 119400 
                 3.6 
                 — 
               
               
                 alone (2100, 3:55, 175) 
               
               
                 20.0 mg/kg GAA + 
                 29775 
                 4.0 
                 185508 
                 190894 
                 4.2 
                 1.60 
               
               
                 100 mg AT2220 
               
               
                 (2100, 3:58, 175) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Baseline- 
                 % Change 
               
               
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
                 subtracted 
                 from 
               
               
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
                 Ratio 
                 Baseline 
               
               
                   
               
               
                 rhGAA Alone 
                 7 
                 844.7 
                   
                   
                 — 
               
               
                 rhGAA + 
                 7 
                 942.0 
                 1.1 
                 — 
                 — 
               
               
                 100 mg AT2220 
               
               
                 Follow-up for 
                 28 
                 N/A 
               
               
                 Baseline 
               
               
                   
               
               
                 Body weight: 105.1 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2029-0203 (Patient H) (Cohort 2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 t½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 20.6 mg/kg GAA alone 
                 17907 
                 4.0 
                 107204 
                 110173 
                 4.6 
                 — 
               
               
                 (1850, 3:42, 175) 
               
               
                 20.6 mg/kg GAA + 
                 23243 
                 4.0 
                 161694 
                 166686 
                 4.4 
                 1.51 
               
               
                 100 mg AT2220 
               
               
                 (1850, 3:44, 175) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Baseline- 
                 % Change 
               
               
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
                 subtracted 
                 from 
               
               
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
                 Ratio 
                 Baseline 
               
               
                   
               
               
                 rhGAA Alone 
                 3 
                 1347.7 
                   
                   
                 54.8 
               
               
                 rhGAA + 
                 3 
                 1103.2 
                 0.8 
                 0.5 
                 26.7 
               
               
                 100 mg AT2220 
               
               
                 Follow-up for 
                 28 
                 870.7 
               
               
                 Baseline 
               
               
                   
               
               
                 Body weight: 89.7 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 6003-0203 (Patient J) (Cohort 2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 t½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 19.9 mg/kg GAA alone 
                 23779 
                 3.0 
                 153974 
                 155601 
                 3.4 
                 — 
               
               
                 (1450, 3:25, 175) 
               
               
                 19.9 mg/kg GAA + 
                 26219 
                 3.0 
                 269271 
                 292137 
                 5.8 
                 1.88 
               
               
                 100 mg AT2220 
               
               
                 (1450, 3:25, 175) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Baseline- 
                 % Change 
               
               
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
                 subtracted 
                 from 
               
               
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
                 Ratio 
                 Baseline 
               
               
                   
               
               
                 rhGAA Alone 
                 3 
                 3908.7 
                   
                   
                 — 
               
               
                 rhGAA + 
                 3 
                 5296.6 
                 1.4 
                 — 
                 — 
               
               
                 100 mg AT2220 
               
               
                 Follow-up for 
                 28 
                 N/A 
               
               
                 Baseline 
               
               
                   
               
               
                 Body weight: 73.0 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
               
                   
               
               
                 rhGAA Activity in Plasma and Muscle for 
               
               
                 Subject 2001-0201 (Patient E) (Cohort 2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Treatment [total dose (mg), 
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                   
                   
               
               
                 duration of infusion (hr:min), 
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 t½ 
               
               
                 infusion rate (mL/hr)] 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 (hr) 
                 F rel   
               
               
                   
               
               
                 GAA alone 
                 26371 
                 4.0 
                 147283 
                 148324 
                 3.13 
                 — 
               
               
                 GAA + 
                 30869 
                 4.0 
                 264841 
                 272353 
                 4.07 
                 1.84 
               
               
                 100 mg AT2220 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Baseline- 
                 % Change 
               
               
                   
                 Biopsy 
                 rhGAA Activity 
                 Fold 
                 subtracted 
                 from 
               
               
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Change 
                 Ratio 
                 Baseline 
               
               
                   
               
               
                 rhGAA Alone 
                 3 
                 1355.4 
                   
                   
                 — 
               
               
                 rhGAA + 
                 3 
                 2138.5 
                 1.6 
                 — 
                 — 
               
               
                 100 mg AT2220 
               
               
                 Follow-up for 
                 28 
                 N/A 
               
               
                 Baseline 
               
               
                   
               
               
                 Body weight: 106.9 kg 
               
            
           
         
       
     
     Table 17 below shows the cumulative AUCs for rhGAA activity in plasma of cohort 2. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Time 
                 2028-0201 
                 2029-0202 
                 6003-0202 
                 2029-0203 
                 6003-0203 
                 2001-0201 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 1472 
                 1769 
                 1443 
                 1610 
                 1034 
                 1068 
                 1220 
                 1337 
                 1671 
                 1659 
                 1386 
                 1454 
               
               
                 2 
                 7965 
                 8315 
                 7828 
                 9621 
                 7705 
                 7937 
                 7214 
                 7450 
                 10499 
                 10615 
                 7949 
                 8983 
               
               
                 3 
                 22987 
                 22573 
                 22121 
                 26716 
                 22685 
                 23428 
                 20011 
                 20154 
                 29546 
                 31020 
                 22185 
                 26097 
               
               
                 4 
                 43259 
                 46080 
                 43930 
                 50207 
                 42426 
                 48006 
                 36988 
                 39704 
                 52442 
                 56689 
                 44429 
                 52571 
               
               
                 5 
                 62103 
                 72142 
                 70971 
                 78427 
                 59273 
                 72180 
                 52355 
                 60508 
                 72064 
                 79937 
                 67125 
                 80667 
               
               
                 6 
                 77708 
                 92769 
                 98881 
                 111294 
                 71064 
                 90597 
                 63550 
                 77746 
                 88561 
                 101224 
                 83577 
                 104121 
               
               
                 7 
                 89164 
                 109551 
                 122363 
                 141376 
                 80584 
                 107070 
                 72339 
                 92388 
                 102076 
                 122069 
                 96025 
                 124866 
               
               
                 8 
                 96948 
                 124505 
                 140329 
                 164267 
                 87328 
                 120678 
                 78879 
                 103874 
                 112225 
                 141195 
                 105784 
                 143990 
               
               
                 9 
                 102833 
                 137830 
                 155433 
                 184205 
                 92657 
                 132007 
                 83646 
                 113265 
                 320241 
                 157686 
                 112846 
                 160285 
               
               
                 10 
                 107669 
                 150103 
                 168382 
                 203675 
                 97345 
                 141786 
                 87638 
                 121326 
                 126846 
                 173053 
                 118271 
                 174339 
               
               
                 12 
                 114263 
                 168912 
                 187295 
                 235455 
                 104183 
                 155687 
                 93505 
                 133617 
                 136582 
                 199017 
                 126701 
                 198053 
               
               
                 24 
                 125868 
                 212384 
                 231174 
                 321065 
                 117942 
                 185508 
                 107204 
                 161694 
                 153974 
                 269271 
                 147283 
                 264841 
               
               
                   
               
            
           
         
       
     
     Table 18A shows rhGAA activity from muscle biopsies taken on Days 3 or 7 of Periods 1 and 2, plus an optional biopsy at follow up for cohort 2 subject. 
     
       
         
           
               
             
               
                 TABLE 18A 
               
             
            
               
                   
               
               
                 rhGAA Activity in Muscle (Cohort 2) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 % Change 
                 Baseline- 
                 Change 
               
               
                   
                 Biopsy 
                 from 
                 subtracted 
                 from 
               
               
                 SubjID/Period 
                 Day 
                 Baseline 
                 Ratio 
                 rhGAA Alone 
               
               
                   
               
               
                 6003-0203* 
                 3 
                 — 
                 — 
                 1.4 
               
               
                 2001-0201* 
                 3 
                 — 
                 — 
                 1.6 
               
               
                 2029-0202/P1 
                 7 
                 178.1 
                 1.9 
                 — 
               
               
                 2029-0202/P2 
                 7 
                 338.6 
                   
                 1.6 
               
               
                 2029-0203/P1 
                 3 
                 54.8 
                 0.5 
                 — 
               
               
                 2029-0203/P2 
                 3 
                 26.7 
                   
                 0.8 
               
               
                 6003-0202* 
                 7 
                 — 
                 — 
                 1.1 
               
               
                 2028-0201/P1 
                 7 
                 13.6 
                 1.4 
                 — 
               
               
                 2028-0201/P2 
                 7 
                 19.2 
                   
                 1.0 
               
               
                   
               
               
                 *Baseline not available 
               
            
           
         
       
     
     Table 18B shows individual muscle 1-DNJ-HCL (AT2220) concentrations taken on Days 3 or 7 of Cohorts 1 and 2. 
     
       
         
           
               
             
               
                 TABLE 18B 
               
             
            
               
                   
               
               
                 Individual Muscle 1-DNJ-HCL (AT2220) Concentrations 
               
               
                 AT2220-010 Muscle AT2220 Conc for Cohort 2 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Muscle AT2220 
               
               
                 SubjID 
                 Dose (mg) 
                 Period 
                 Study Day 
                 Conc (ng/g) a   
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Patient A 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 11.8 
               
               
                 Patient B 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 10.0 
               
               
                 Patient C 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 13.1 
               
               
                 Patient D 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 BLQ 
               
               
                 Patient E 
                 100 
                 mg 
                 Period 2 
                 Day 3 
                 17.8 
               
               
                 Patient F 
                 100 
                 mg 
                 Period 2 
                 Day 7 
                  BLQ b   
               
               
                 Patient F 
                 0 
                 mg 
                 Follow up 
                 Baseline 
                 BLQ 
               
               
                 Patient G 
                 100 
                 mg 
                 Period 2 
                 Day 7 
                 BLQ 
               
               
                 Patient G 
                 0 
                 mg 
                 Follow up 
                 Baseline 
                 BLQ 
               
               
                 Patient H 
                 100 
                 mg 
                 Period 2 
                 Day 3 
                  BLQ b   
               
               
                 Patient H 
                 0 
                 mg 
                 Follow up 
                 Baseline 
                 BLQ 
               
               
                 Patient I 
                 100 
                 mg 
                 Period 2 
                 Day 7 
                  BLQ b   
               
               
                 Patient J 
                 100 
                 mg 
                 Period 2 
                 Day 3 
                  BLQ c   
               
               
                   
               
               
                   a LLOQ = 8.0 ng/g 
               
               
                   b Due to small muscle volume, sample was diluted 2-fold, therefore LLOQ = 16.0 ng/g 
               
               
                   c Due to small muscle volume, sample was diluted 3-fold, therefore LLOQ = 32.0 ng/g 
               
            
           
         
       
     
     Summary of Results 
     Single doses of 50 mg and 100 mg 1-DNJ-HCl (AT2220) have been found safe and well-tolerated in patients with Pompe Disease. Only mild, transient adverse events (AE&#39;s) have been reported, none of which were related to AT2220 (representative adverse events are described below). One serious AE was citalopram-induced QTc prolongation. The QTc prolongation attenuated following citalopram dose reduction. Generally, urine hex 4 levels either did not change from baseline, or did not show any consistent trend following a single dose of AT2220 ( FIG. 7 ). Additionally, CPK levels did not appreciably change from baseline in Cohorts 1 and 2 ( FIG. 8 ). 
     Plasma rhGAA activity AUC increased for all patients for both co-administered doses relative to alglucosidase alfa alone. Increases in AUC were primarily driven by prolonged plasma half-life due to increases in rhGAA activity at post-Tmax time points (Table 10A,  FIG. 2A  and  FIG. 4 ). The increases in plasma rhGAA activity AUC suggest an increase in stabilized rhGAA uptake for tissue distribution. 
     Muscle biopsies were taken on Day 7 for all four Cohort 1 patients, and on Day 3 or Day 7 for each of 3 of the 6 Cohort 2 patients. Three patients from Cohort 2 had an optional Day 30 muscle biopsy, that was used as a baseline for those patients. Of the Cohort 1 patients, following co-administration of 50 mg AT2220, one had a 40% increase in muscle rhGAA activity, two showed no change, and one had a 30% decrease in rhGAA activity relative to rhGAA alone. Of the Cohort 2 patients, following co-administration of 100 mg AT2220, two patients had 60% and 40% increases in rhGAA activity relative to rhGAA alone, but one was decreased by 20% from biopsies taken on Day 3. From biopsies taken on Day 7, two were increased by 60% and 10%, and one showed no change in rhGAA activity. 
     The pharmacokinetics of plasma AT2220 are approximately linear for the 50 mg and 100 mg doses evaluated at this point in the study. An approximate 2-fold increase in Cmax and AUC is observed with dose (Table 10B,  FIG. 5 ). The rate of absorption (Tmax) is 2 to 3 hours indicating all bioavailable drug has been absorbed early on during the infusion of rhGAA. Muscle AT2220 concentrations from Day 3 or Day 7 biopsies were either below or near the lower limit of quantitation of 8 ng/g (Table 18B). 
     Total plasma rhGAA protein by Western blot followed a similar trend as plasma rhGAA activity in terms of AT2220 dose-related increases ( FIG. 6 , Table 10C). 
     CONCLUSIONS 
     1-DNJ-HCl was safe and well-tolerated at both 50 mg and 100 mg dose levels evaluated to date. 
     Plasma rhGAA activity increased 20% to 40% and 50% to 90% following single dose of 50 mg and 100 mg 1-DNJ-HCl, respectively. 
     At the 50 mg dose level, 1 of 4 patients had increased rhGAA activity in muscle; however, at 100 mg 1-DNJ-HCl, 4 of 6 patients had up to 60% increases in rhGAA activity in muscle. 
     Plasma 1-DNJ-HCl demonstrated approximately linear PK for the 2 doses evaluated to date. 
     1-DNJ-HCl concentrations in muscle were either below or just above the lower limit of quantification from Day 3 or 7 biopsies suggesting 1-DM-HCl may not accumulate following multiple dosing every 14 days. 
     Plasma total rhGAA protein PK followed a similar trend to rhGAA activity PK. 
     Adverse Events 
     20 adverse events (AEs) have been reported, one of which was serious. Representative adverse events are shown in Tables 19 and 20. The serious AE, prolonged QTc interval from 473 to 493 msec, which occurred after the screening visit, but prior to dosing, was moderate in severity, and was considered unrelated to study drug by the investigator. All other AEs were mild in severity, all considered unrelated to study drug, and resolved without treatment. Urine hexose tetrasaccharide A (urine Hex 4) and serum CPK levels are presented for each patient in  FIGS. 7 and 8 . 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Cohort 1: Adverse Events 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Investigator 
                   
                   
                   
                   
                 Any 
                 Relation 
                 Was 
                   
               
               
                 No./Patient 
                 Adverse 
                   
                   
                   
                 Treatment 
                 to Study 
                 Event 
               
               
                 No. 
                 Event 
                 Start Date 
                 Stop Date 
                 Outcome 
                 Required? 
                 Drug? 
                 Serious? 
                 Intensity 
               
               
                   
               
               
                 20310101 
                 SINUS 
                 31 JAN. 2012 
                 04 FEB. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 BLOCKED 
                   
                   
                 Resolved 
               
               
                   
                 WITH 
               
               
                   
                 RUNNY 
               
               
                   
                 NOSE 
               
               
                   
                 TIRED, 
                 31 JAN. 2012 
                 04 FEB. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 MORE THAN 
                   
                   
                 Resolved 
               
               
                   
                 USUAL 
               
               
                 20310102 
                 BILATERAL 
                 06 JAN. 2012 
                 31 JAN. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 THIGH PAIN 
                   
                   
                 Resolved 
               
               
                   
                 FREQUENCY 
                 06 JAN. 2012 
                 09 JAN. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 OF 
                   
                   
                 Resolved 
               
               
                   
                 URINATION 
               
               
                   
                 INCREASED 
               
               
                   
                 TO APPROX 
               
               
                   
                 HOURLY. 
               
               
                   
                 LONGER 
                 06 JAN. 2012 
                 09 JAN. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 SLEEP 
                   
                   
                 Resolved 
               
               
                   
                 INCREASED 
               
               
                   
                 NAP 
               
               
                   
                 FREQUENCY 
               
               
                   
                 R LEG SORE 
                 10 JAN. 2012 
                   
                 Recovering/ 
                 Concomitant 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 “SHOOTING 
                   
                   
                 Resolving 
                 Medications 
               
               
                   
                 PAIN” WHEN 
               
               
                   
                 PRESSURE 
               
               
                   
                 APPLIED TO 
               
               
                   
                 SITE 
               
               
                   
                 RIGHT 
                 05 JAN. 2012 
                 09 JAN. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 INTERIOR 
                   
                   
                 Resolved 
               
               
                   
                 SIDE OF 
               
               
                   
                 MOUTH 
               
               
                   
                 FELT LIKE 
               
               
                   
                 TISSUE WAS 
               
               
                   
                 CHEWED ON 
               
               
                   
                 UPPER 
                 06 JAN. 2012 
                 09 JAN. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 RESPIRATORY/ 
                   
                   
                 Resolved 
               
               
                   
                 FLU LIKE 
               
               
                   
                 SYMPTOMS 
               
               
                 20310103 
                 PAIN IN 
                 05 JAN. 2012 
                 20 JAN. 2012 
                 Recovered/ 
                 Concomitant 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 RIGHT 
                   
                   
                 Resolved 
                 Medications 
               
               
                   
                 THIGH 
               
               
                 60030101 
                 ACHING 
                 25 NOV. 2011 
                 25 NOV. 2011 
                 Recovered/ 
                 Concomitant 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 FEET 
                   
                   
                 Resolved 
                 Medications 
               
               
                   
               
            
           
         
       
         
         
           
             Safety data reviewed for 4 subjects 
             No SAE or drug-related AEs 
             Urine Hex4: no drug related elevations 
             ALT/AST: No drug related elevations 
             CPK: no drug related elevations beyond subjects&#39; baseline values range—no clinically significant changes 
             Muscle strength: No change between Periods 1 and 2 
             Follow-up occurs 24-30 days after Period 2, Visit 4
           3 subjects showed elevation in Hex4 and CPK levels   Additional follow-up showed all 3 subjects had Hex4 and CPK levels within baseline range   changes were consistent with background noise   
         
           
         
       
    
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Cohort 2: Adverse Events 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Investigator 
                   
                   
                   
                   
                 Any 
                 Relation 
                 Was 
                   
               
               
                 No./Patient 
                   
                   
                   
                   
                 Treatment 
                 to Study 
                 Event 
               
               
                 No. 
                 Adverse Event 
                 Start Date 
                 Stop Date 
                 Outcome 
                 Required? 
                 Drug? 
                 Serious? 
                 Intensity 
               
               
                   
               
               
                 60030202 
                 None 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 20280201 
                 RIGHT LEG 
                 21 MAR. 2012 
                 22 MAR. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 PAIN POST 
                   
                   
                 Resolved 
               
               
                   
                 MUSCLE 
               
               
                   
                 BIOPSY 
               
               
                 20290202 
                 None 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                 20290203 
                 ERYTHEMA 
                 05 APR. 2012 
                 07 APR. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 AT PORT SITE 
                   
                   
                 Resolved 
               
               
                   
                 SPREADING 
               
               
                   
                 TO HALFWAY 
               
               
                   
                 UP NECK AND 
               
               
                   
                 MID-CHEST 
               
               
                   
                 DUE TO 
               
               
                   
                 ALLERGIC 
               
               
                   
                 REACTION TO 
               
               
                   
                 TAPE 
               
               
                   
                 TENDERNESS 
                 06 APR. 2012 
                 19 APR. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 AT MUSCLE 
                   
                   
                 Resolved 
               
               
                   
                 BIOPSY SITE 
               
               
                   
                 RIGHT LEG 
               
               
                   
                 BRUISING AT 
                 06 APR. 2012 
                 17 APR. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 MUSCLE 
                   
                   
                 Resolved 
               
               
                   
                 BIOPSY SITE 
               
               
                   
                 RIGHT LEG 
               
               
                   
                 ERYTHEMA 
                 19 APR. 2012 
                 21 APR. 2012 
                 Recovered/ 
                 None 
                 Unrelated 
                 No 
                 Mild 
               
               
                   
                 AT PORT SITE 
                   
                   
                 Resolved 
               
               
                   
                 DUE TO 
               
               
                   
                 ALLERGIC 
               
               
                   
                 REACTION TO 
               
               
                   
                 TAPE 
               
               
                 60030203 
                 None 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
               
            
           
         
       
         
         
           
             Safety data reviewed for 5 subjects 
             No SAE or drug-related AEs 
             Hex4: no drug related elevations 
             ALT/AST: No drug related elevations 
             CPK: no drug related elevations beyond subjects&#39; baseline range 
             Muscle strength: No change between Periods 1 and 2 
           
         
       
    
     Example 3 
     Dosing Regimen for the Treatment of Pompe Disease Using 1-Deoxynojirimycin Hydrochloride and Alglucosidase Alfa—Cohorts 3 and 4 
     One objective of the study was to evaluate the safety, effectiveness, and pharmacodynamics of dose regimens comprising co-administering 1-deoxynojirimycin hydrochloride and alglucosidase alfa in patients with Pompe disease. 
     Another objective of the study was to assess the effects of various doses of 1-deoxynojirimycin hydrochloride on the GAA activity. This was evaluated by measuring the GAA enzyme activity and protein levels in skeletal muscle at Day 3 and/or Day 7 following a single intravenous infusion with alglucosidase alfa alone and after pre-administration of single ascending oral doses of 1-deoxynojirimycin hydrochloride. 
     Methods. 
     The study was conducted essentially according to the methods described in Example 1. Cohort 3 comprised 6 subjects. Cohort 4 comprised 7 subjects. Each subject received alglucosidase alfa alone as an intravenous infusion in Period 1 and a single 250 mg dose (Cohort 3) or 600 mg (Cohort 4) of 1-deoxynojirimycin hydrochloride one hour before initiation of an intravenous infusion of alglucosidase alfa in Period 2. 
     Results: Cohort 3 
     Plasma and Muscle rhGAA Activity Increases with Co-Administration of 1-Deoxynojirimycin Hydrochloride and Acid α-Glucosidase Relative to Acid α-Glucosidase Alone 
     For co-administration of acid α-glucosidase with 250 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), a 2.0-fold mean increase in plasma rhGAA activity was observed, as shown in Table 21. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Plasma rhGAA Activity PK Summary for Cohorts 1, 2, and 3. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 C max   a   
                   
                 AUC 0-T   a   
                 AUC 0-∞   a   
                   
                   
               
               
                 Period/ 
                   
                   
                 (nmol/ 
                 T max   b   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 
                 t½ c   
               
               
                 Treatment 
                 Cohort 
                 Period 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio a   
                 (hr) 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 1 
                 16441 (31.6) 
                 5.0 (4-5) 
                 105834 (30.7) 
                 107707 (31.1) 
                 — 
                 3.9 (0.6) 
               
               
                 (N = 4) 
               
               
                 rhGAA + 
                 1 
                 2 
                 19922 (24.4) 
                 5.0 (4-6) 
                 152207 (21.3) 
                 157162 (22.4) 
                 1.5 (13.2) 
                 4.2 (0.8) 
               
               
                 50 mg AT2220 
               
               
                 (N = 4) 
               
               
                 rhGAA Alone 
                 2 
                 1 
                 22785 (18.0) 
                 4.0 (3-5) 
                 142415 (30.4) 
                 144953 (31.5) 
                 — 
                 3.8 (0.6) 
               
               
                 (N = 6) 
               
               
                 rhGAA + 
                 2 
                 2 
                 28607 (13.9) 
                 4.0 (3-6) 
                 229434 (25.3) 
                 241203 (27.5) 
                 1.7 (10.1) 
                 4.9 (0.8) 
               
               
                 100 mg AT2220 
               
               
                 (N = 6) 
               
               
                 rhGAA Alone 
                 3 
                 1 
                 18986 (18.7) 
                 4.0 (4-4) 
                 107774 (19.7) 
                 109165 (20.0) 
                   
                 3.6 (0.5) 
               
               
                 (N = 6) 
               
               
                 rhGAA + 
                 3 
                 2 
                 22651 (9.0)  
                 4.0 (4-4) 
                 201224 (8.8)  
                 214751 (9.5)  
                 2.0 (19.71 
                 5.3 (1.1) 
               
               
                 250 mg AT2220 
               
               
                 (N = 6) 
               
               
                   
               
               
                   a Geometric mean (CV %) 
               
               
                   b Median (range) 
               
               
                   c Arithmetic mean (SD) 
               
            
           
         
       
     
     For co-administration of acid α-glucosidase with 250 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), increases in plasma rhGAA activity of cohort 3 subjects are shown in Tables 22-35. These tables also show the increase in rhGAA in muscle tissue following co-administration of acid α-glucosidase with 250 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1). 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 rhGAA Activity in Plasma for Subject 2027-0301. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                 Treatment (Total Dose [mg], 
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Duration of Infusion [hr:min]) 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
               
                 20.0 mg/kg rhGAA Alone 
                 2027-0301 
                 22352 
                 4.0 
                 112800 
                 113502 
                 — 
                 3.2 
               
               
                 (1540, 3:41) 
               
               
                 20.0 mg/kg rhGAA + 
                 2027-0301 
                 23309 
                 4.0 
                 187370 
                 192337 
                 1.7 
                 4.0 
               
               
                 250 mg AT2220 
               
               
                 (1540, 3:55) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 rhGAA Activity in Plasma for Subject 2029-0304. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                 Treatment (Total Dose [mg], 
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Duration of Infusion [hr:min]) 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
               
                 19.9 mg/kg rhGAA Alone 
                 2029-0304 
                 23813 
                 4.0 
                 134941 
                 136708 
                 — 
                 3.7 
               
               
                 (1900, 3:44) 
               
               
                 19.9 mg/kg rhGAA + 
                 2029-0304 
                 23628 
                 4.0 
                 209939 
                 225285 
                 16 
                 5.7 
               
               
                 250 mg AT2220 (1900, 4:02) 
               
               
                   
               
               
                 Body Weight = 95.6 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 rhGAA Activity in Plasma for Subject 2029-0305. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                 Treatment (Total Dose [mg], 
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Duration of Infusion [hr:min]) 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
               
                 20.5 mg/kg rhGAA Alone 
                 2029-0305 
                 19996 
                 4.0 
                 115349 
                 117667 
                 — 
                 4.3 
               
               
                 (1400, 3:44) 
               
               
                 20.5 mg/kg rhGAA + 
                 2029-0305 
                 25898 
                 4.0 
                 233726 
                 248494 
                 2.1 
                 5.3 
               
               
                 250 mg AT2220 
               
               
                 (1400, 3:49) 
               
               
                   
               
               
                 Body Weight = 68.3 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 rhGAA Activity in Plasma for Subject 2029-0306. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                 Treatment (Total Dose [mg], 
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Duration of Infusion [hr:min]) 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/ml) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 20.4 mg/kg rhGAA Alone 
                 2029-0306 
                 14548 
                 4.0 
                 97198 
                 98713 
                 — 
                 3.7 
               
               
                 (1300, 3:43) 
               
               
                 20.4 mg/kg rhGAA + 
                 2029-0306 
                 21631 
                 4.0 
                 189711 
                 215411 
                 2.2 
                 7.2 
               
               
                 250 mg AT2220 
               
               
                 (1300, 3:59) 
               
               
                   
               
               
                 Body Weight = 63.8 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 rhGAA Activity in Plasma for Subject 2029-0307. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                 Treatment (Total Dose [mg], 
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Duration of Infusion [hr:min]) 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/ml) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 19.9 mg/kg rhGAA Alone 
                 2029-0307 
                 15758 
                 4.0 
                 73754 
                 74024 
                 — 
                 2.9 
               
               
                 (1200, 3:43) 
               
               
                 19.9 mg/kg rhGAA + 
                 2029-0307 
                 21983 
                 4.0 
                 189648 
                 196051 
                 2.6 
                 4.3 
               
               
                 250 mg AT2220 
               
               
                 (1200, 3:43) 
               
               
                   
               
               
                 Body Weight = 60.4 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 rhGAA Activity in Plasma for Subject 6003-0304. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                 Treatment (Total Dose [mg], 
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Duration of Infusion [hr:min]) 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/ml) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
               
                 20.2 mg/kg rhGAA Alone 
                 6003-0304 
                 19195 
                 4.0 
                 124504 
                 126855 
                 — 
                 3.8 
               
               
                 (1200, 4:00) 
               
               
                 20.2 mg/kg rhGAA + 
                 6003-0304 
                 19912 
                 4.0 
                 200698 
                 215705 
                 1.7 
                 5.5 
               
               
                 250 mg AT2220 
               
               
                 (1200, 3:50) 
               
               
                   
               
               
                 Body Weight = 59.5 kg 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 PK Table: individual subjects and statistical summary for cohort 3. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                   
                   
                   
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Cohort 
                 Period 
                 Treatment 
                 SubjID 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Cohort 3 
                 Period 1 
                 rhGAA 
                 2027-0301 
                 22352 
                 4.0 
                 112800 
                 113502 
                 — 
                 3.2 
               
               
                   
                   
                 Alone 
                 2029-0304 
                 23813 
                 4.0 
                 134941 
                 136708 
                 — 
                 3.7 
               
               
                   
                   
                   
                 2029-0305 
                 19996 
                 4.0 
                 115349 
                 117667 
                 — 
                 4.3 
               
               
                   
                   
                   
                 2029-0306 
                 14548 
                 4.0 
                 97198 
                 98713 
                 — 
                 3.7 
               
               
                   
                   
                   
                 2029-0307 
                 15758 
                 4.0 
                 73754 
                 74024 
                 — 
                 2.9 
               
               
                   
                   
                   
                 6003-0304 
                 19195 
                 4.0 
                 124504 
                 126855 
                 — 
                 3.8 
               
               
                   
                   
                   
                 N 
                 6 
                 6 
                 6 
                 6 
                 — 
                 6 
               
               
                   
                   
                   
                 Mean 
                 19277 
                 4.0 
                 109758 
                 111245 
                 — 
                 3.6 
               
               
                   
                   
                   
                 SD 
                 3614 
                 0.0 
                 21662 
                 22275 
                 — 
                 0.5 
               
               
                   
                   
                   
                 Min 
                 14548 
                 4.0 
                 73754 
                 74024 
                 — 
                 2.9 
               
               
                   
                   
                   
                 Median 
                 19596 
                 4.0 
                 114075 
                 115585 
                 — 
                 3.7 
               
               
                   
                   
                   
                 Max 
                 23813 
                 4.0 
                 134941 
                 136708 
                 — 
                 4.3 
               
               
                   
                   
                   
                 CV % 
                 18.7 
                 0.0 
                 19.7 
                 20.0 
                 — 
                 13.6 
               
               
                   
                   
                   
                 Geometric 
                 18986 
                 4.0 
                 107774 
                 109165 
                 — 
                 3.6 
               
               
                   
                   
                   
                 Mean 
               
               
                 Cohort 3 
                 Period 2 
                 rhGAA + 
                 2027-0301 
                 23309 
                 4.0 
                 187370 
                 192337 
                 1.7 
                 4.0 
               
               
                   
                   
                 250 mg 
                 2029-0304 
                 23628 
                 4.0 
                 209939 
                 225285 
                 1.6 
                 5.7 
               
               
                   
                   
                 AT2220 
                 2029-0305 
                 25898 
                 4.0 
                 233726 
                 248494 
                 2.1 
                 5.3 
               
               
                   
                   
                   
                 2029-0306 
                 21631 
                 4.0 
                 189711 
                 215411 
                 2.2 
                 7.2 
               
               
                   
                   
                   
                 2029-0307 
                 21983 
                 4.0 
                 189648 
                 196051 
                 2.6 
                 4.3 
               
               
                   
                   
                   
                 6003-0304 
                 19912 
                 4.0 
                 200698 
                 215705 
                 1.7 
                 5.5 
               
               
                   
                   
                   
                 N 
                 6 
                 6 
                 6 
                 6 
                 6 
                 6 
               
               
                   
                   
                   
                 Mean 
                 22727 
                 4.0 
                 201849 
                 215547 
                 2.0 
                 5.3 
               
               
                   
                   
                   
                 SD 
                 2044 
                 0.0 
                 17826 
                 20490 
                 0.39 
                 1.1 
               
               
                   
                   
                   
                 Min 
                 19912 
                 4.0 
                 187370 
                 192337 
                 1.6 
                 4.0 
               
               
                   
                   
                   
                 Median 
                 22646 
                 4.0 
                 195205 
                 215558 
                 1.9 
                 5.4 
               
               
                   
                   
                   
                 Max 
                 25898 
                 4.0 
                 233726 
                 248494 
                 2.6 
                 7.2 
               
               
                   
                   
                   
                 CV % 
                 9.0 
                 0.0 
                 8.8 
                 9.5 
                 19.7 
                 21.4 
               
               
                   
                   
                   
                 Geometric 
                 22651 
                 4.0 
                 201224 
                 214751 
                 2.0 
                 5.2 
               
               
                   
                   
                   
                 Mean 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Cumulative Plasma rhGAA Activity AUCs for cohort 3. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 2027-0301 
                 2029-0304 
                 2029-0305 
                 2029-0306 
                 2029-0307 
                 6003-0304 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
                 Period 1 
                 Period 2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 1 
                 1298 
                 1133 
                 1267 
                 1143 
                 1220 
                 1393 
                 792 
                 1010 
                 1062 
                 1215 
                 909 
                 1301 
               
               
                 2 
                 7879 
                 7326 
                 8113 
                 7960 
                 7497 
                 8969 
                 5424 
                 5326 
                 5868 
                 8077 
                 5949 
                 6683 
               
               
                 3 
                 21394 
                 21155 
                 22525 
                 22418 
                 21222 
                 26305 
                 15301 
                 15140 
                 16684 
                 23310 
                 16839 
                 18909 
               
               
                 4 
                 40802 
                 41579 
                 43265 
                 43017 
                 39887 
                 50408 
                 28612 
                 32464 
                 31637 
                 43887 
                 33196 
                 36732 
               
               
                 5 
                 59642 
                 64183 
                 63849 
                 64625 
                 56227 
                 74955 
                 41909 
                 51551 
                 44153 
                 63736 
                 50697 
                 55908 
               
               
                 6 
                 72995 
                 84032 
                 79311 
                 84019 
                 68144 
                 95856 
                 53007 
                 67542 
                 52334 
                 80847 
                 64811 
                 72918 
               
               
                 7 
                 — 
                 100016 
                 91047 
                 100716 
                 77913 
                 114242 
                 62208 
                 81827 
                 58134 
                 96913 
                 75545 
                 88421 
               
               
                 8 
                 89787 
                 113607 
                 100193 
                 115598 
                 85835 
                 130563 
                 69700 
                 94724 
                 62129 
                 111305 
                 84044 
                 102543 
               
               
                 9 
                 95144 
                 124902 
                 107586 
                 129048 
                 91696 
                 144498 
                 75249 
                 106837 
                 64856 
                 122982 
                 91391 
                 115053 
               
               
                 10 
                 99116 
                 134855 
                 113075 
                 140047 
                 96015 
                 156950 
                 79526 
                 117553 
                 66898 
                 133094 
                 97158 
                 126666 
               
               
                 12 
                 104289 
                 151892 
                 120450 
                 158424 
                 102185 
                 177830 
                 85692 
                 134802 
                 69635 
                 150378 
                 105787 
                 146551 
               
               
                 24 
                 112800 
                 187370 
                 134941 
                 209939 
                 115349 
                 233726 
                 97198 
                 189711 
                 73754 
                 189648 
                 124504 
                 200698 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 30 
               
               
                   
               
               
                 Cumulative Plasma rhGAA Activity AUCs 0-Tmax and Tmax-12 h for cohorts 1-3. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Cohort 1: Period 1 rhGAA Alone, Period 2 rhGAA + 50 mg AT2220 
                   
               
            
           
           
               
               
               
               
            
               
                 Time 
                 2031-0101 
                 2031-0102 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 5.0 
                 5.0 
                 0.0 
                 0.0 
                 5.0 
                 6.0 
                 1.0 
                 20 
               
               
                 AUC 0-Tmax   
                 43537 
                 46504 
                 2967 
                 6.8 
                 52486 
                 68442 
                 15955 
                 30.4 
               
               
                 AUC Tmax-12 h   
                 96302 
                 133401 
                 37099 
                 38.5 
                 109601 
                 138339 
                 28739 
                 26.2 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Time 
                 2031-0103 
                 6003-0101 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 5.0 
                 5.0 
                 0.0 
                 0.0 
                 4.0 
                 4.0 
                 0.0 
                 0.0 
               
               
                 AUC 0-Tmax   
                 29815 
                 37448 
                 7632 
                 25.6 
                 43327 
                 43801 
                 474 
                 1.1 
               
               
                 AUC Tmax-12 h   
                 61036 
                 92644 
                 31609 
                 51.8 
                 113716 
                 142712 
                 28997 
                 25.5 
               
               
                   
               
            
           
           
               
            
               
                 Cohort 2: Period 1 rhGAA Alone, Period 2 rhGAA + 100 mg AT2220 
               
            
           
           
               
               
               
            
               
                 Time 
                 2028-0201 
                 2029-0202 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 4.0 
                 4.0 
                 0.0 
                 0.0 
                 5.0 
                 6.0 
                 1.0 
                 20.0 
               
               
                 AUC 0-Tmax   
                 43259 
                 46080 
                 2821 
                 6.5 
                 70971 
                 111294 
                 40323 
                 56.8 
               
               
                 AUC Tmax-12 h   
                 114263 
                 168912 
                 54649 
                 47.8 
                 187295 
                 235455 
                 48160 
                 25.7 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Time 
                 6003-0202 
                 2029-0203 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 4.0 
                 4.0 
                 0.0 
                 0.0 
                 4.0 
                 4.0 
                 0.0 
                 0.0 
               
               
                 AUC 0-Tmax   
                 42426 
                 48006 
                 5580 
                 13.2 
                 36988 
                 39704 
                 2716 
                 7.3 
               
               
                 AUC Tmax-12 h   
                 104183 
                 155687 
                 51504 
                 49.4 
                 93505 
                 133617 
                 40112 
                 42.9 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Time 
                 6003-0203 
                 2001-0201 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 3.0 
                 3.0 
                 0.0 
                 0.0 
                 4.0 
                 4.0 
                 0.0 
                 0.0 
               
               
                 AUC 0-Tmax   
                 29546 
                 31020 
                 1474 
                 5.0 
                 44429 
                 52571 
                 8141 
                 18.3 
               
               
                 AUC Tmax-12 h   
                 136582 
                 199017 
                 62435 
                 45.7 
                 126701 
                 198053 
                 71352 
                 56.3 
               
               
                   
               
            
           
           
               
            
               
                 Cohort 3: Period 1 rhGAA Alone, Period 2 rhGAA + 250 mg AT2220 
               
            
           
           
               
               
               
            
               
                 Time 
                 2027-0301 
                 2029-0304 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 4.0 
                 4.0 
                 0.0 
                 0.0 
                 4.0 
                 4.0 
                 0.0 
                 0.0 
               
               
                 AUC 0-Tmax   
                 40802 
                 41579 
                 777 
                 1.9 
                 43265 
                 43017 
                 −248 
                 −0.6 
               
               
                 AUC Tmax-12 h   
                 104289 
                 151892 
                 47603 
                 45.6 
                 120450 
                 158424 
                 37974 
                 31.5 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Time 
                 2029-0305 
                 2029-0306 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 4.0 
                 4.0 
                 0.0 
                 0.0 
                 4.0 
                 4.0 
                 0.0 
                 0.0 
               
               
                 AUC 0-Tmax   
                 39887 
                 50408 
                 10521 
                 26.4 
                 28612 
                 32464 
                 3852 
                 13.5 
               
               
                 AUC Tmax-12 h   
                 102185 
                 177830 
                 75644 
                 74.0 
                 85692 
                 134802 
                 49110 
                 57.3 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Time 
                 2029-0307 
                 6003-0304 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (hr) 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
                 Period 1 
                 Period 2 
                 Difference 
                 % Change 
               
               
                   
               
               
                 T max   
                 4.0 
                 4.0 
                 0.0 
                 0.0 
                 4.0 
                 4.0 
                 0.0 
                 0.0 
               
               
                 AUC 0-Tmax   
                 31637 
                 43887 
                 12250 
                 38.7 
                 33196 
                 36732 
                 3537 
                 10.7 
               
               
                 AUC Tmax-12 h   
                 69635 
                 150378 
                 80743 
                 116.0 
                 105787 
                 146551 
                 40764 
                 38.5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Mean (SD) Plasma AT2220 Profile for Cohorts 1 (N = 4), 2 (N = 6), and 3 (N = 6). 
               
               
                 AT2220 PK Summary Table 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 C max   a   
                 T max   b   
                 AUC 0-T   a   
                 AUC 0-∞   a   
                 AUC 0-∞   
                 t½ c   
               
               
                 AT2220 Dose 
                 (ng/mL) 
                 (hr) 
                 (ng · hr/mL) 
                 (ng · hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
               
                  50 mg (N = 4) 
                 1035 (25.0) 
                 2.0 (1-3) 
                  6972 (28.1) 
                  7008 (28.2) 
                 — 
                 3.1 (0.2) 
               
               
                 100 mg (N = 6) 
                 1863 (13.1) 
                 3.0 (1-4) 
                 13003 (18.9) 
                 13137 (19.2) 
                 1.9 
                 3.4 (0.3) 
               
               
                 250 mg (N = 6) 
                 3150 (32.5) 
                 2.0 (2-3) 
                 21760 (19.8) 
                 22083 (19.2) 
                 3.2 
                 3.7 (0.6) 
               
               
                   
               
               
                   a Geometric mean (CV %) 
               
               
                   b Median (range) 
               
               
                   c Arithmetic mean (SD) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Plasma AT2220 PK Summary Table for Cohort 3. 
               
               
                 AT2220-010 Cohort 3 Plasma AT2220 PK Parameters 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 AT2220 
                   
                 C max   
                 T max   
                 AUC 0-T   
                 AUC 0-∞   
                 T½ 
               
               
                 Cohort 
                 Dose 
                 SubjID 
                 (ng/mL) 
                 (hr) 
                 (ng*hr/mL) 
                 (ng*hr/mL) 
                 (hr) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Cohort 3 
                 250 mg 
                 2027-0301 
                 3620 
                 2.0 
                 22230 
                 22345 
                 3.0 
               
               
                   
                   
                 2029-0304 
                 2350 
                 2.0 
                 18321 
                 18711 
                 4.1 
               
               
                   
                   
                 2029-0305 
                 4220 
                 2.0 
                 24278 
                 24558 
                 3.8 
               
               
                   
                   
                 2029-0306 
                 2090 
                 3.0 
                 18707 
                 19329 
                 4.6 
               
               
                   
                   
                 2029-0307 
                 2740 
                 2.0 
                 19344 
                 19534 
                 3.4 
               
               
                   
                   
                 6003-0304 
                 4750 
                 3.0 
                 29670 
                 29916 
                 3.4 
               
               
                   
                   
                 N 
                 6 
                 6 
                 6 
                 6 
                 6 
               
               
                   
                   
                 Mean 
                 3295 
                 2.3 
                 22092 
                 22399 
                 3.7 
               
               
                   
                   
                 SD 
                 1071 
                 0.5 
                 4369 
                 4299 
                 0.6 
               
               
                   
                   
                 Min 
                 2090 
                 2.0 
                 18321 
                 18711 
                 3.0 
               
               
                   
                   
                 Median 
                 3180 
                 2.0 
                 20787 
                 20940 
                 3.6 
               
               
                   
                   
                 Max 
                 4750 
                 3.0 
                 29670 
                 29916 
                 4.6 
               
               
                   
                   
                 CV % 
                 32.5 
                 22.1 
                 19.8 
                 19.2 
                 15.4 
               
               
                   
                   
                 Geometric 
                 3150 
                 2.3 
                 21760 
                 22083 
                 3.7 
               
               
                   
                   
                 Mean 
               
               
                   
               
            
           
         
       
         
         
           
             Mean Cmax and AUC increased in an approximately dose-proportional manner (1.8- and 1.9-fold, respectively) from 50 mg to 100 mg. 
             However, Cmax and AUC increased in a less than dose-proportional manner to 250 mg (3.0- and 3.2-fold, respectively). 
           
         
       
    
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Muscle rhGAA Activity Summary of Cohorts 1-3. 
               
               
                 AT2220-010 Muscle rhGAA Activity All Cohorts 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Muscle rhGAA 
                   
                 % Change 
                 % Change 
               
               
                   
                   
                   
                   
                 Biopsy 
                 Activity 
                 P2/P1 
                 from 
                 from 
               
               
                 SubjID 
                 Cohort 
                 Period 
                 Treatment 
                 Day 
                 (pmol/mp/hr) 
                 Ratio 
                 Period 1 
                 Baseline 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 2031-0101 
                 Cohort 1 
                 Period 1 
                 rhGAA Alone 
                 7 
                 1352.8 
                   
                   
                   
               
               
                 2031-0101 
                 Cohort 1 
                 Period 2 
                 rhGAA + 50 mg AT2220 
                 7 
                 1398.5 
                 1.0 
                 3.4 
               
               
                 2031-0102 
                 Cohort 1 
                 Period 1 
                 rhGAA Alone 
                 7 
                 1010.5 
               
               
                 2031-0102 
                 Cohort 1 
                 Period 2 
                 rhGAA + 50 mg AT2220 
                 7 
                 956.3 
                 0.95 
                 −5.4 
               
               
                 2031-0103 
                 Cohort 1 
                 Period 1 
                 rhGAA Alone 
                 7 
                 988.4 
               
               
                 2031-0103 
                 Cohort 1 
                 Period 2 
                 rhGAA + 50 mg AT2220 
                 7 
                 1383.1 
                 1.4 
                 39.9 
               
               
                 6003-0101 
                 Cohort 1 
                 Period 1 
                 rhGAA Alone 
                 7 
                 1620.4 
               
               
                 6003-0101 
                 Cohort 1 
                 Period 2 
                 rhGAA + 50 mg AT2220 
                 7 
                 1109.0 
                 0.68 
                 −31.6 
               
               
                 2028-0201 
                 Cohort 2 
                 Period 1 
                 rhGAA Alone 
                 7 
                 756.4 
                   
                   
                 13.6 
               
               
                 2028-0201 
                 Cohort 2 
                 Period 2 
                 rhGAA + 100 mg AT2220 
                 7 
                 794.1 
                 1.0 
                 5.0 
                 19.2 
               
               
                 2028-0201 
                 Cohort 2 
                 Follow up 
                 Baseline 
                 28 
                 666.0 
               
               
                 2029-0202 
                 Cohort 2 
                 Period 1 
                 rhGAA Alone 
                 7 
                 1019.9 
                   
                   
                 178 
               
               
                 2029-0202 
                 Cohort 2 
                 Period 2 
                 rhGAA + 100 mg AT2220 
                 7 
                 1608.9 
                 1.6 
                 57.8 
                 339 
               
               
                 2029-0202 
                 Cohort 2 
                 Follow up 
                 Baseline 
                 28 
                 366.8 
               
               
                 2029-0203 
                 Cohort 2 
                 Period 1 
                 rhGAA Alone 
                 3 
                 1347.7 
                   
                   
                 54.8 
               
               
                 2029-0203 
                 Cohort 2 
                 Period 2 
                 rhGAA + 100 mg AT2220 
                 3 
                 1103.2 
                 0.82 
                 −18.1 
                 26.7 
               
               
                 2029-0203 
                 Cohort 2 
                 Follow up 
                 Baseline 
                 28 
                 870.7 
               
               
                 6003-0202 
                 Cohort 2 
                 Period 1 
                 rhGAA Alone 
                 7 
                 844.7 
               
               
                 6003-0202 
                 Cohort 2 
                 Period 2 
                 rhGAA + 100 mg AT2220 
                 7 
                 942.0 
                 1.1 
                 11.5 
               
               
                 6003-0203 
                 Cohort 2 
                 Period 1 
                 rhGAA Alone 
                 3 
                 3908.7 
               
               
                 6003-0203 
                 Cohort 2 
                 Period 2 
                 rhGAA + 100 mg AT2220 
                 3 
                 5296.6 
                 1.4 
                 35.5 
               
               
                 2001-0201 
                 Cohort 2 
                 Period 1 
                 rhGAA Alone 
                 3 
                 1355.4 
               
               
                 2001-0201 
                 Cohort 2 
                 Period 2 
                 rhGAA + 100 mg AT2220 
                 3 
                 2138.5 
                 1.6 
                 57.8 
               
               
                 2027-0301 
                 Cohort 3 
                 Period 1 
                 rhGAA Alone 
                 7 
                 BLQ 
               
               
                 2027-0301 
                 Cohort 3 
                 Period 2 
                 rhGAA + 250 mg AT2220 
                 7 
                 2090.7 
                 ∞ 
                 ∞ 
               
               
                 2029-0304 
                 Cohort 3 
                 Period 1 
                 rhGAA Alone 
                 7 
                 1350.2 
               
               
                 2029-0304 
                 Cohort 3 
                 Period 2 
                 rhGAA + 250 mg AT2220 
                 7 
                 1322.0 
                 1.0 
                 −2.1 
               
               
                 2029-0305 
                 Cohort 3 
                 Period 1 
                 rhGAA Alone 
                 7 
                 937.9 
               
               
                 2029-0305 
                 Cohort 3 
                 Period 2 
                 rhGAA + 250 mg AT2220 
                 7 
                 1746.6 
                 1.9 
                 86.2 
               
               
                 2029-0306 
                 Cohort 3 
                 Period 1 
                 rhGAA Alone 
                 3 
                 2934.9 
                   
                   
                 1100 
               
               
                 2029-0306 
                 Cohort 3 
                 Period 2 
                 rhGAA + 250 mg AT2220 
                 3 
                 2819 
                 1.0 
                 −3.9 
                 1053 
               
               
                 2029-0306 
                 Cohort 3 
                 Follow up 
                 Baseline 
                 28 
                 244.5 
               
               
                 2029-0307 
                 Cohort 3 
                 Period 1 
                 rhGAA Alone 
                 3 
                 2228.9 
               
               
                 2029-0307 
                 Cohort 3 
                 Period 2 
                 rhGAA + 250 mg AT2220 
                 3 
                 1883.9 
                 0.85 
                 −15.5 
               
               
                 6003-0304 
                 Cohort 3 
                 Period 1 
                 rhGAA Alone 
                 3 
                 2755 
               
               
                 6003-0304 
                 Cohort 3 
                 Period 2 
                 rhGAA + 250 mg AT2220 
                 3 
                 3896.9 
                 1.4 
                 41.4 
               
               
                 Mean 
                 Cohort 1 
                   
                   
                 Day 7 
                 N = 4 
                 1.0 
                 1.6 
               
               
                 Mean 
                 Cohort 2 
                   
                   
                 Day 3 
                 N = 3 
                 1.3 
                 25.0 
               
               
                 Mean 
                 Cohort 2 
                   
                   
                 Day 7 
                 N = 3 
                 1.2 
                 24.8 
               
               
                 Mean 
                 Cohort 3 
                   
                   
                 Day 3 
                 N = 3 
                 1.1 
                 7.3 
               
               
                 Mean 
                 Cohort 3 
                   
                   
                 Day 7 
                 N = 2 
                 1.4 
                 42.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 AT2220 Concentrations in muscle from biopsies taken 3 
               
               
                 or 7 days post dose in cohorts 1-3. 
               
               
                 AT2220-010 Muscle AT2220 Conc for Cohorts 1, 2, and 3 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Co- 
                   
                   
                   
                 Muscle AT2220 
               
               
                 SubjID 
                 hort 
                 Dose (mg) 
                 Period 
                 Study Day 
                 Conc (ng/g) a   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 2031-0101 
                 1 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 11.8 
               
               
                 2031-0102 
                 1 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 10.0 
               
               
                 2031-0103 
                 1 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 13.1 
               
               
                 6003-0101 
                 1 
                 50 
                 mg 
                 Period 2 
                 Day 7 
                 BLQ b   
               
               
                 2001-0201 
                 2 
                 100 
                 mg 
                 Period 2 
                 Day 3 
                 17.8 
               
               
                 2028-0201 
                 2 
                 100 
                 mg 
                 Period 2 
                 Day 7 
                 BLQ b   
               
               
                 2028-0201 
                 2 
                 0 
                 mg 
                 Follow up 
                 Baseline 
                 BLQ  
               
               
                 2029-0202 
                 2 
                 100 
                 mg 
                 Period 2 
                 Day 7 
                 BLQ  
               
               
                 2029-0202 
                 2 
                 0 
                 mg 
                 Follow up 
                 Baseline 
                 BLQ  
               
               
                 2029-0203 
                 2 
                 100 
                 mg 
                 Period 2 
                 Day 3 
                 BLQ c   
               
               
                 2029-0203 
                 2 
                 0 
                 mg 
                 Follow up 
                 Baseline 
                 BLQ  
               
               
                 6003-0202 
                 2 
                 100 
                 mg 
                 Period 2 
                 Day 7 
                 BLQ b   
               
               
                 6003-0203 
                 2 
                 100 
                 mg 
                 Period 2 
                 Day 3 
                 BLQ c   
               
               
                 2027-0301 
                 3 
                 250 
                 mg 
                 Period 2 
                 Day 7 
                 ISV d   
               
               
                 2029-0304 
                 3 
                 250 
                 mg 
                 Period 2 
                 Day 7 
                 28.1 
               
               
                 2029-0305 
                 3 
                 250 
                 mg 
                 Period 2 
                 Day 7 
                 27.9 
               
               
                 2029-0306 
                 3 
                 250 
                 mg 
                 Period 2 
                 Day 3 
                 BLQ  
               
               
                 2029-0306 
                 3 
                 0 
                 mg 
                 Follow up 
                 Baseline e   
                 BLQ c   
               
               
                 2029-0307 
                 3 
                 250 
                 mg 
                 Period 2 
                 Day 3 
                 54.4 
               
               
                 6003-0304 
                 3 
                 250 
                 mg 
                 Period 2 
                 Day 3 
                 61.9 
               
               
                   
               
               
                   a LLOQ = 8.0 ng/g 
               
               
                   b Due to small muscle volume, sample was diluted 2-fold, therefore LLOQ = 16.0 ng/g 
               
               
                   c Due to small muscle volume, sample was diluted 3-fold, therefore LLOQ = 32.0 ng/g 
               
               
                   d No result due to insufficient sample for assay 
               
               
                   e 29 days since AT2220 administration 
               
            
           
         
       
         
         
           
             Preliminary muscle AT2220 concentrations are available from biopsies taken 3 or 7 days post dose and at follow up for baseline 
             4 of 6 Cohort 3 subjects had a quantifiable AT2220 concentration in muscle. 
             Dose-related increases in Cohort 3 muscle AT2220 concentrations are observed. 
             Increased Cohort 3 muscle AT2220 concentrations are also study day related. 
           
         
       
    
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 rhGAA Activity in PBMCs: Summary of Cohorts 1-3. 
               
               
                 Arithmetic Mean 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Period 2/Period 1 
                 % Change from 
               
               
                 Cohort 
                 N 
                 Time 
                 Ratio 
                 Period 1 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Cohort 1 
                 4 
                 0 
                 2.6 
                 157 
               
               
                 Cohort 1 
                 4 
                 4 h 
                 0.8 
                 −17.5 
               
               
                 Cohort 1 
                 4 
                 Day 2 
                 0.9 
                 −13.9 
               
               
                 Cohort 1 
                 4 
                 Day 7 
                 1.0 
                 4.1 
               
               
                 Cohort 2 
                 6 
                 0 
                 0.9 
                 −12.4 
               
               
                 Cohort 2 
                 6 
                 4 h 
                 0.9 
                 −8.4 
               
               
                 Cohort 2 
                 6 
                 Day 2 
                 1.8 
                 81.0 
               
               
                 Cohort 2 
                 3 
                 Day 3 
                 0.6 
                 −36.7 
               
               
                 Cohort 2 
                 3 
                 Day 7 
                 0.9 
                 −5.9 
               
               
                 Cohort 3 
                 6 
                 0 
                 2.1 
                 107 
               
               
                 Cohort 3 
                 6 
                 4 h 
                 11.3 
                 1028 
               
               
                 Cohort 3 
                 6 
                 Day 2 
                 1.5 
                 47.5 
               
               
                 Cohort 3 
                 3 
                 Day 3 
                 3.4 
                 240 
               
               
                 Cohort 3 
                 3 
                 Day 7 
                 1.5 
                 49.0 
               
               
                   
               
            
           
         
       
     
     Results: Cohort 4 
     Plasma and Muscle rhGAA Activity Increases with Co-Administration of 1-Deoxynojirimycin Hydrochloride and Acid α-Glucosidase Relative to Acid α-Glucosidase Alone 
     For co-administration of acid α-glucosidase with 600 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1), increases in plasma rhGAA activity of cohort 4 subjects are shown in Tables 36-38. These tables also show the increase in rhGAA in muscle tissue following co-administration of acid α-glucosidase with 600 mg 1-deoxynojirimycin hydrochloride (Period 2) relative to ERT alone (Period 1). 
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 PK Table: individual subjects and statistical summary of plasma rhGAA activity in cohort 4. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 C max   
                   
                 AUC 0-T   
                 AUC 0-∞   
                 P2/P1 
                   
               
               
                   
                   
                   
                   
                 (nmol/ 
                 T max   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 0-∞   
                 t½ 
               
               
                 Treatment 
                 Period 
                 SubjID 
                 Cohort 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio 
                 (hr) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 rhGAA 
                 Period 1 
                 2033-0402 
                 Cohort 4 
                 29604 
                 4.0 
                 196222 
                 203729 
                 — 
                 4.7 
               
               
                 Alone 
                   
                 2304-0401 
                 Cohort 4 
                 23192 
                 4.0 
                 157216 
                 159700 
                 — 
                 3.9 
               
               
                   
                   
                 2304-0402 
                 Cohort 4 
                 11314 
                 3.0 
                 59064 
                 59320 
                 — 
                 3.1 
               
               
                   
                   
                 2008-0101 
                 Cohort 4 
                 15760 
                 4.0 
                 105281 
                 106938 
                 — 
                 3.6 
               
               
                   
                   
                 2030-0401 
                 Cohort 4 
                 25506 
                 4.0 
                 169773 
                 175606 
                 — 
                 4.7 
               
               
                   
                   
                 2304-0403 
                 Cohort 4 
                 18693 
                 4.0 
                 103687 
                 103890 
                 — 
                 2.6 
               
               
                   
                   
                 6003-0405 
                 Cohort 4 
                 13337 
                 6.0 
                 91510 
                 93071 
                 — 
                 3.5 
               
               
                   
                   
                   
                 N 
                 7 
                 7 
                 7 
                 7 
                 7 
                 7 
               
               
                   
                   
                   
                 Mean 
                 19629 
                 4.1 
                 126108 
                 128893 
                 — 
                 3.7 
               
               
                   
                   
                   
                 SD 
                 6724 
                 0.9 
                 49022 
                 51579 
                 — 
                 0.8 
               
               
                   
                   
                   
                 Min 
                 11314 
                 3.0 
                 59064 
                 59320 
                 — 
                 2.6 
               
               
                   
                   
                   
                 Median 
                 18693 
                 4.0 
                 105281 
                 106938 
                 — 
                 3.6 
               
               
                   
                   
                   
                 Max 
                 29604 
                 6.0 
                 196222 
                 203729 
                 — 
                 4.7 
               
               
                   
                   
                   
                 CV % 
                 34.3 
                 21.7 
                 38.9 
                 40.0 
                 — 
                 20.9 
               
               
                   
                   
                   
                 Geometric 
                 18628 
                 4.1 
                 117489 
                 119620 
                 — 
                 3.7 
               
               
                   
                   
                   
                 Mean 
               
               
                 rhGAA + 
                 Period 2 
                 2033-0402 
                 Cohort 4 
                 30226 
                 5.0 
                 297583 
                 341719 
                 1.7 
                 7.3 
               
               
                 600 mg 
                   
                 2304-0401 
                 Cohort 4 
                 31820 
                 4.0 
                 359201 
                 427452 
                 2.7 
                 8.5 
               
               
                 AT2220 
                   
                 2304-0402 
                 Cohort 4 
                 15145 
                 4.0 
                 152737 
                 167209 
                 2.8 
                 6.3 
               
               
                   
                   
                 2008-0101 
                 Cohort 4 
                 17421 
                 5.0 
                 190273 
                 215315 
                 2.0 
                 6.7 
               
               
                   
                   
                 2030-0401 
                 Cohort 4 
                 34985 
                 4.0 
                 345028 
                 390285 
                 2.2 
                 7.0 
               
               
                   
                   
                 2304-0403 
                 Cohort 4 
                 24031 
                 3.0 
                 203896 
                 208703 
                 2.0 
                 4.0 
               
               
                   
                   
                 6003-0405 
                 Cohort 4 
                 13704 
                 6.0 
                 141283 
                 157166 
                 1.7 
                 6.2 
               
               
                   
                   
                   
                 N 
                 7 
                 7 
                 7 
                 7 
                 7 
                 7 
               
               
                   
                   
                   
                 Mean 
                 23905 
                 4.4 
                 241429 
                 272550 
                 2.2 
                 6.6 
               
               
                   
                   
                   
                 SD 
                 8643 
                 1.0 
                 90992 
                 111358 
                 0.4 
                 1.4 
               
               
                   
                   
                   
                 Min 
                 13704 
                 3.0 
                 141283 
                 157166 
                 1.7 
                 4.0 
               
               
                   
                   
                   
                 Median 
                 24031 
                 4.0 
                 203896 
                 215315 
                 2.0 
                 6.7 
               
               
                   
                   
                   
                 Max 
                 34985 
                 6.0 
                 359201 
                 427452 
                 2.8 
                 8.5 
               
               
                   
                   
                   
                 CV % 
                 36.2 
                 22.0 
                 37.7 
                 40.9 
                 20.8 
                 20.9 
               
               
                   
                   
                   
                 Geometric 
                 22505 
                 4.3 
                 226809 
                 253526 
                 2.1 
                 6.4 
               
               
                   
                   
                   
                 Mean 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 PK Summary Table: All Cohorts (1-4). 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 C max   a   
                   
                 AUC 0-T   a   
                 AUC 0-∞   a   
                   
                   
               
               
                 Period/ 
                   
                   
                 (nmol/ 
                 T max   b   
                 (hr*nmol/ 
                 (hr*nmol/ 
                 AUC 
                 t½ c   
               
               
                 Treatment 
                 Cohort 
                 Period 
                 hr/mL) 
                 (hr) 
                 hr/mL) 
                 hr/mL) 
                 Ratio a   
                 (hr) 
               
               
                   
               
               
                 rhGAA Alone 
                 1 
                 1 
                 16441 (31.6) 
                 5.0 (4-5) 
                 105834 (30.7) 
                 107707 (31.1) 
                 — 
                 3.9 (0.6) 
               
               
                 (N = 4) 
               
               
                 rhGAA + 
                 1 
                 2 
                 19922 (24.4) 
                 5.0 (4-6) 
                 152207 (21.3) 
                 157162 (22.4) 
                 1.5 (13.2) 
                 4.2 (0.8) 
               
               
                 50 mg AT2220 
               
               
                 (N = 4) 
               
               
                 rhGAA Alone 
                 2 
                 1 
                 22785 (18.0) 
                 4.0 (3-5) 
                 142415 (30.4) 
                 144953 (31.5) 
                 — 
                 3.8 (0.6) 
               
               
                 (N = 6) 
               
               
                 rhGAA + 
                 2 
                 2 
                 28607 (13.9) 
                 4.0 (3-6) 
                 229434 (25.3) 
                 241203 (27.5) 
                 1.7 (10.1) 
                 4.9 (0.8) 
               
               
                 100 mg AT2220 
               
               
                 (N = 6) 
               
               
                 rhGAA Alone 
                 3 
                 1 
                 18986 (18.7) 
                 4.0 (4-4) 
                 107774 (19.7) 
                 109165 (20.0) 
                 — 
                 3.6 (0.5) 
               
               
                 (N = 6) 
               
               
                 rhGAA + 
                 3 
                 2 
                 22651 (9.0)  
                 4.0 (4-4) 
                 201224 (8.8)  
                 214751 (9.5)  
                 2.0 (19.7) 
                 5.3 (1.1) 
               
               
                 250 mg AT2220 
               
               
                 (N = 6) 
               
               
                 rhGAA Alone 
                 4 
                 1 
                 18628 (34.3) 
                 4.0 (3-6) 
                 117489 (38.9) 
                 119620 (40.0) 
                 — 
                 3.7 (0.8) 
               
               
                 (N = 7) 
               
               
                 rhGAA + 
                 4 
                 2 
                 22505 (36.2) 
                 4.0 (3-6) 
                 226809 (37.7) 
                 253526 (40.9) 
                 2.1 (20.8) 
                 6.6 (1.4) 
               
               
                 600 mg AT2220 
               
               
                 (N = 7) 
               
               
                   
               
               
                   a Geometric mean (CV %) 
               
               
                   b Median (range) 
               
               
                   c Arithmetic mean (SD) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 AT2220-010 Muscle rhGAA Activity Summary of Cohort 4 Subjects. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Muscle rhGAA 
                   
                   
               
               
                   
                   
                   
                   
                 Biopsy 
                 Activity 
                 P2/P1 
                 Tx/BSL 
               
               
                 SubjID 
                 Cohort 
                 Period 
                 Treatment 
                 Day 
                 (pmol/mg/hr) 
                 Ratio 
                 Ratio 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 2033-0402 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 3 
                 1020.0 
                   
                 2.2 
               
               
                 2033-0402 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 3 
                 3052.7 
                 3.0 
                 6.7 
               
               
                 2033-0402 
                 Cohort 4 
                 Follow up 
                 Baseline 
                 28  
                 454.8 
               
               
                 2304-0401 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 7 
                 1179.6 
               
               
                 2304-0401 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 7 
                 1261.2 
                 1.1 
               
               
                 2304-0402 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 7 
                 901.6 
                   
                 1.2 
               
               
                 2304-0402 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 7 
                 991.1 
                 1.1 
                 1.3 
               
               
                 2304-0402 
                 Cohort 4 
                 Follow up 
                 Baseline 
                 28  
                 765.1 
               
               
                 2008-0101 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 3 
                 2262.2 
                   
                 2.2 
               
               
                 2008-0101 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 3 
                 3785.9 
                 1.7 
                 3.6 
               
               
                 2008-0101 
                 Cohort 4 
                 Follow up 
                 Baseline 
                 Follow-up 
                 1047.3 
               
               
                 2030-0401 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 3 
                 
                   a 
                 
               
               
                 2030-0401 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 3 
                 1722.3 
                 N/A 
               
               
                 2304-0403 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 3 
                 
                   b 
                 
                   
                 N/A 
               
               
                 2304-0403 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 3 
                 
                   b 
                 
                 N/A 
                 N/A 
               
               
                 2304-0403 
                 Cohort 4 
                 Follow up 
                 Baseline 
                 Follow-up 
                 
                   b 
                 
               
               
                 6003-0405 
                 Cohort 4 
                 Period 1 
                 rhGAA Alone 
                 7 
                 723.6 
               
               
                 6003-0405 
                 Cohort 4 
                 Period 2 
                 rhGAA + 600 mg AT2220 
                 7 
                 1021.3 
                 1.4 
               
               
                   
               
               
                   a  Muscle activty could not be accurately determined due to low protein levels from a presumed fatty tissue sample 
               
               
                   b  Biopsy samples mislabed at the site; could not be confirmed 
               
            
           
         
       
     
     Summary of Results 
     For people with Pompe disease, deficient GAA enzyme leads to the accumulation of glycogen in tissues affected by disease (primarily muscle). Preclinical data (Khanna et al. PLoS ONE (2012) 7(7): e40776. doi:10.1371/journal.pone.0040776) demonstrated that AT2220 in combination with ERT enhances rhGAA enzyme activity, reduces glycogen accumulation, and potentially mitigates ERT-related immunogenicity in a mouse model of Pompe disease. In the present study described by Examples 1-3, co-administration of AT2220 to Pompe patients increased rhGAA enzyme activity and enhanced rhGAA enzyme uptake into muscle tissue compared to ERT alone. 
     The study described by Examples 1-3 is a Phase 2 open-label, multi-center study to evaluate the safety and PK effects of four increasing oral doses of AT2220 (50 mg (Cohort 1), 100 mg (Cohort 2), 250 mg (Cohort 3), or 600 mg (Cohort 4)) co-administered with ERT (Myozyme®/Lumizyme®) versus ERT alone in males and females with Pompe disease. The study enrolled male and female patients who had been on a stable dose and regimen of ERT for at least three months. All patients were given a regularly scheduled ERT infusion. One hour prior to the initiation of the next ERT infusion, patients received a single oral dose of AT2220. Plasma rhGAA activity and protein levels were evaluated during each infusion. Each patient underwent muscle biopsies three or seven days after each infusion to measure tissue GAA enzyme activity with and without the chaperone, as well as to measure the level of AT2220 in the muscle. 
     Safety: Single doses of AT2220 co-administered with ERT were well-tolerated, with no drug-related adverse events reported. In addition, AT2220 was cleared from muscle to near-undetectable levels by Day 7 in all four cohorts. 
     Recombinant Human GAA (rhGAA) Enzyme Activity in Plasma: 24-hour plasma pharmacokinetics (PK) was measured during and after each infusion. Plasma rhGAA activity increased in 23 out of 23 patients (100%) following co-administration and the increases were dose-related. These data suggest that co-administration increases the amount of stabilized, properly folded, and active rhGAA enzyme available for uptake into tissue. Table 39 and  FIG. 21  show a summary of rhGAA enzyme activity in plasma area under curve (AUC) for cohorts 1-4 described by Examples 1-3. 
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 rhGAA enzyme activity in plasma area under curve (AUC) for cohorts 1-4. 
               
               
                 rhGAA Enzyme Activity in Plasma Area Under Curve (AUC) 
               
               
                 ERT + AT2220 vs. ERT Alone 
               
            
           
           
               
               
               
            
               
                   
                 Cohort 
                 % Increase vs. ERT Alone 
               
               
                   
                   
               
               
                   
                 1 (n = 4) 
                  50% 
               
               
                   
                 2 (n = 6) 
                  70% 
               
               
                   
                 3 (n = 6) 
                 100% 
               
               
                   
                 4 (n = 7) 
                 110% 
               
               
                   
                   
               
            
           
         
       
     
     Enzyme Activity in Muscle: 
     Muscle biopsies were taken to measure GAA enzyme uptake into muscle tissue, with and without AT2220. In Cohort 1, all 4 patients had muscle biopsies on Day 7. In Cohorts 2-4, muscle biopsies were taken on Day 3 for half the patients, and on Day 7 for the other half of patients. 
     In Cohort 1, no consistent change in GAA enzyme activity was observed at day 7. In Cohorts 2, 3, and 4 the results show that more enzyme is taken up into muscle tissue following AT2220 co-administration compared to ERT alone. The effect was most pronounced at the highest (600 mg) dose of AT2220. Table 40 and  FIG. 22  show a summary of GAA. enzyme activity in muscle at day 3 for cohorts 2-4 described by Examples 1-3. 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 GAA enzyme activity in muscle at day 3 for cohorts 2-4. 
               
               
                 GAA Enzyme Activity in Muscle at Day 3 
               
               
                 ERT + AT2220 vs. ERT Alone 
               
            
           
           
               
               
               
            
               
                   
                 Cohort 
                 % Increase vs. ERT Alone 
               
               
                   
                   
               
               
                   
                 2 (n = 3) 
                 25% 
               
               
                   
                 3 (n = 3) 
                  7% 
               
               
                   
                 4 (n = 2) 
                 133%  
               
               
                   
                   
               
            
           
         
       
     
     At Day 3 the GAA enzyme activity in muscle following co-administration compared to ERT alone in patients with evaluable biopsies increased by the following: 25% in Cohort 2 (n=3), 7% in Cohort 3 (n=3), and 133% in Cohort 4 (n=2). At Day 7 the GAA enzyme activity in muscle was lower relative to Day 3, as expected based on the cellular half-life of the enzyme. However, following co-administration compared to ERT alone in patients with evaluable biopsies the following increases were sustained: 20% in Cohort 2 (n=3), 40% in Cohort 3 (n=2), and 20% in Cohort 4 (n=3). 
     Effect of AT2220 on ERT-Related Immunogenicity Measured Ex Vivo: 
     By stabilizing the folded and active form of the rhGAA enzyme, AT2220 may mitigate ERT-induced immunogenicity since unfolded and aggregated proteins are generally more antigenic than properly folded proteins. Recent published studies show that approximately 40% of the administered ERT can be captured by circulating antibodies and infusion associated reactions occur in approximately 50% of Pompe patients receiving ERT infusions (Banati et al., Muscle Nerve. 2011 November; 44(5):720-6). Initial ex vivo studies using T cells derived from blood from 50 healthy donors demonstrated that the addition of AT2220 may significantly reduce the immunogenicity of Myozyme® and Lumizyme®. The studies utilized Antitope Ltd.&#39;s EpiScreen™ assay and are being repeated in samples from the Pompe patients in the study described by Examples 1-3. 
     Example 4 
     Skeletal Muscle Distribution and Half-Life of N-Butyl-DNJ (AT2221) is Similar to 1-DNJ (AT2220) 
     Eight-week old wild type C57BL/6 mice where administered an oral dose of 100 mg/kg of 1-DNJ or N-butyl-DNJ. Plasma and tissue samples were taken at 0.5, 2, 4, 24, 48, 72, 96, 120, 144, &amp; 168 hours post administration and the presence of drug was analyzed. The concentration of drug in plasma is expressed as ng/ml. The concentration of drug in tissue samples is expressed as ng/g. 
     As shown in  FIG. 23 , the skeletal muscle distribution and half-life of N-butyl-DNJ (AT2221) is similar to 1-DNJ (AT2220). The Cmax of AT2220 was 120 uM. The Cmax of AT2221 was 140 uM. 
     Example 5 
     N-Butyl-DNJ (AT2221) and 1-DNJ (AT2220) have a Similar Effect on Phramacokinetics of rhGAA 
     Eight week old GAA KO mice were administered rhGAA (10 mg/kg IV). Oral AT2220 or AT2221 (100 mg/kg) was administered 30 min prior to GAA (Myozyme); Plasma samples were taken at pre-dose, 0.08, 0.25, 0.50, 0.75, 1, 2, 4, 8, and 24 hours after administration of GAA and the enzyme activity was determined. 
     As shown in  FIG. 24 , AT2220 and AT2221 increased the circulating half-life of rhGAA by at least ˜2-fold. N-butyl-DNJ (AT2221) and 1-DNJ (AT2220) have a similar effect on Phramacokinetics of rhGAA. 
     A Western blot of recombinant GAA in plasma at 2, 8, and 24 hours after i.v. administration of GAA is shown in  FIG. 25  for mice administered rhGAA with or without AT2220 or AT2221. 
     Example 6 
     Coadministration of DNJ or NB-DNJ with rhGAA has a Similar Effect on Glycogen Reduction 
     Twelve week old GAA KO mice were administered 20 mg/kg i.v. recombinant human GAA (Myozyme) every other week for 8 weeks. An oral dose of AT2220 or AT2221 (30 mg/kg) was administered 30 minutes prior to rhGAA (Myozyme). Tissues were collected 21 days after the last dose of rhGAA and the level of glycogen (GAA substrate) was measured. 
     As shown in  FIG. 26 , coadministration of DNJ or NB-DNJ with rhGAA has a similar effect on glycogen reduction. n=5-mice/group; *p&lt;0.05 vs. untreated t-test; #p&lt;0.05 vs. Myozyme alone t-test; Dotted line indicates wild type glycogen levels. Cmax ˜40 μM following administration of 30 mg/kg AT2220 or AT2221; equivalent to approximately 600 mg in humans. 
     Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.