WO2017049161A1 - ACID ALPHA-GLUCOSIDASE AND A β-2 AGONIST FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDER - Google Patents

Abstract

The present disclosure is directed to methods of treating a lysosomal storage disorder by administering a therapeutic agent as an adjunctive therapy to lysosomal enzyme replacement therapy. Other embodiments are directed to methods of increasing expression of a receptor for a lysosomal enzyme and methods of treating a lysosomal storage disorder. Some embodiments are directed to a method of treating a glycogen storage disease II in an individual by administering to the individual an acid alpha-glucosidase and a β2 agonist. Some embodiments herein are directed to a method of treating a glycogen storage disease II by administering (1) rhGAA; and (2) clenbuterol.

Description

Acid Alpha-Glucosidase and a β-2 Agonist for the Treatment

of a Lysosomal Storage Disorder

Cross-Reference to Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/220,751 filed September 18, 2015, the disclosure of which is incorporated by reference herein in its entirety.

Summary

[0002] Embodiments herein are directed to a method of treating a lysosomal storage disorder in an individual in need thereof, the method comprising administering to the individual a therapeutic agent as an adjunctive therapy to lysosomal enzyme replacement therapy. Some embodiments herein are directed to a method of increasing expression of a receptor for a lysosomal enzyme, the method comprising administering a therapeutic agent as an adjunctive therapy to an individual who is undergoing or has undergone lysosomal enzyme replacement therapy. Some embodiments herein are directed to a method of treating a lysosomal storage disorder in an individual in need thereof, the method comprising administering (1) a lysosomal enzyme; and (2) another therapeutic agent. Some embodiments herein are directed to a method of treating a glycogen storage disease II in an individual in need thereof, the method comprising administering (1) an acid alpha-glucosidase; and (2) a β2 agonist. Some embodiments are directed to a method of treating a lysosomal storage disorder comprising administering a composition comprising a lysosomal enzyme and another therapeutic agent. Some embodiments are directed to a composition comprising a lysosomal enzyme and a therapeutic agent.

[0003] In some embodiments, the lysosomal storage disorder may be glycogen storage disease II.

[0004] In some embodiments, the lysosomal enzyme may be acid alpha- glucosidase (acid a-glucosidase or GAA). In some embodiments, the other therapeutic agent is a beta-2-adrenergic agonist (β2 agonist).

[0005] In some embodiments, the β2 agonist is a selective β2 agonist. In some embodiments, the β2 agonist is clenbuterol, albuterol, formoterol, salmeterol, or a combination thereof. In some embodiments, the acid a-glucosidase may be GAA, rhGAA, nco-rhGAA, reveglucosidase alpha, alglucosidase alfa, an rhGAA with higher M6P content than naturally occurring GAA that is administered with a chaperone (e.g. 1- deoxynojirimycin), or a combination thereof. In some embodiments, the lysosomal enzyme is acid alpha-glucosidase. In some embodiments, the other therapeutic agent is a β2 agonist. Some embodiments are directed to a method of treating glycogen storage disease II comprising administering a composition comprising a β2 agonist and an acid alpha- glucosidase.

[0006] In some embodiments, the lysosomal storage disorder is characterized by reduced or deficient activity of a lysosomal enzyme. In some embodiments, the lysosomal storage disorder to be treated is characterized by reduced or deficient activity of the lysosomal enzyme in the brain of the patient. In certain embodiments, the lysosomal enzyme that is deficient in a patient to be treated is acid a-glucosidase.

[0007] In some embodiments, the lysosomal storage disorder may be Pompe disease (glycogen storage disease II ("GSD II")), Gaucher disease, Fabry disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, or Niemann-Pick disease. In some embodiments, the lysosomal storage disorder is glycogen storage disease II (Pompe disease). In some embodiments, the glycogen storage disease II may be late-onset Pompe disease ("LOPD"), juvenile-onset Pompe disease, or infantile Pompe disease.

Description of the Figures

[0008] Fig. 1 illustrates that enzyme replacement therapy (ERT) depends upon receptor- mediated uptake of recombinant lysosomal enzymes. Fig. 1(A) illustrates that in lysosomal storage disorders the CI-MPR is expressed at low levels on the cell membrane, and therefore a drug that increased CI-MPR would enhance biochemical correction from ERT. Fig. 1(B) illustrates that a selective p2-agonist, clenbuterol, increased CI-MPR expression and significantly enhanced biochemical correction in combination with ERT, in comparison with ERT alone, as demonstrated by decreased glycogen storage in mice with a classical lysosomal storage disorder, Pompe disease.

[0009] Fig. 2 illustrates the use of urinary Glc₄ as a biomarker in mice with Pompe disease. GAA-KO mice were administered four weekly doses of rhGAA (20 mg/kg), and treated with clenbuterol (6 mg/L in drinking water), albuterol (30 mg/L in drinking water), ERT alone, or untreated (Mock). Urinary Glc₄ was analyzed after 4 weeks of therapy. Mean +/- standard deviation are shown. Statistically significant alterations associated with clenbuterol treatment indicated (** = p<0.001).

[0010] Fig. 3 illustrates a natural history study of LOPD. Sixteen subjects with LOPD were evaluated following enrollment in a research protocol approved by the Duke University IRB. Fig. 3(A) illustrates muscle testing with handheld dynamometry was performed on all subjects. Strength relative to normal controls is shown. Fig. 3(B) illustrates pulmonary function testing was available for 12 subjects. Mean +/- s.d. shown, relative to normal controls.

[0011] Fig. 4 illustrates the study design and efficacy of a 24-week study of adjunctive albuterol. Fig 4A illustrates the study design, indicating timing for study visits when patients were seen, telephone visits, electrocardiograms (EKG), and pulmonary function tests (PFT). Fig. 4B illustrates the 6 minute walk test ("6MWT") distance at the indicated study visits. Each line connects the datapoints for one research subject. Right (Fig. 4C) and left (Fig. 4D) hand grip strength tested by dynamometry.

[0012] Fig. 5 illustrates the comparison of urinary Glc₄ biomarker in LOPD at baseline and after 12 weeks. Urinary Glc₄ for a treated (ERT) group (n=57) and a placebo group (n=28) is represented. Bar and lines represent baseline and 12-week means and standard deviations. Paired T-test analysis showed a significant decrease (p=0.0002) in Glc₄ excretion for the treated group after 12 weeks of ERT, but not for the placebo group over the same time.

[0013] Fig. 6 illustrates the biochemical correction of striated muscle following AAV vector administration and adjunctive small molecule therapy in heart, diaphragm and quadriceps: (A) levels of GAA, and (B) glycogen content. Mean +/- SEM is shown. P<0.05 (*),. P<0.01 (**), and PO.OOl (***).

[0014] Fig. 7 illustrates results of Muscle Function Testing, showing (A) Latency change in wirehang testing for each treatment group; and (B) change in body weight. Mean +/- SEM is shown. P<0.05 (*),. P<0.01 (**), and PO.001 (***). [0015] Fig. 8 illustrates the effect of the small molecules alone. To evaluate drug effect in biochemical levels, several different muscles were collected to perform (A) GAA assay and (B) Glycogen content assay for heart, as well as (C) wirehang testing. Mean +/- SEM is shown. P<0.05 (*),. P<0.01 (**), and PO.OOl (***).

[0016] Fig. 9 illustrates the effect of small molecules upon CI-MPR and LC3. Western blotting and quantification for CI-MPR and LC3-II in (A) heart and (B) quadriceps (n=5 in each group). The signals for CI-MPR and for LC3-II were normalized to tubulin (heart) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Mean +/- SEM is shown. P<0.05 (*),. P<0.01 (**), and PO.OOl (***).

Detailed Description

[0017] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0018] The lysosomal storage disorders (LSDs) are characterized by deficiencies in lysosomal enzymes resulting from mutations in genes that encode the enzyme proteins and related cofactors. Lysosomal enzymes degrade most biomolecules, the products of which are then recycled in a process that is essential for cell health and growth. LSDs result in accumulation of undegraded products in lysosomes and concomitant cell enlargement, dysfunction, and death. The clinical manifestations of LSDs include neuronal lipidosis, leukodystrophy, mucopolysaccharidosis, and storage histiocytosis. [0019] Pompe disease (Glycogen storage disease type II; acid maltase deficiency; MTM 232300) is a myopathy, similar to limb-girdle muscular dystrophy in its late-onset form, which results from acid a-glucosidase (GAA) deficiency in striated and smooth muscle. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) has been effective in a subset of patients with Pompe disease. Infantile-onset Pompe disease affects the heart and skeletal muscle primarily, and causes death early in childhood from cardiorespiratory failure related to an underlying hypertrophic cardiomyopathy, if initiation of ERT is delayed or the patient fails to respond sustainably due to high, sustained anti-GAA antibodies.

[0020] GAA normally functions as an acid hydrolase that metabolizes lysosomal glycogen, and GAA deficiency causes lysosomal glycogen accumulation in virtually all tissues. The availability of ERT with rhGAA has prolonged survival and ameliorated the cardiomyopathy of infantile Pompe disease. In late-onset Pompe disease ERT has largely resulted in stabilization of the disease process from a pulmonary and motor perspective. Documented limitations of ERT in Pompe disease include the requirement for frequent intravenous infusions of high levels of GAA to achieve efficacy, degree of pre-ERT muscle damage, and the possibility of humoral immunity. The rhGAA doses are markedly higher than doses required for ERT in other lysosomal storage disorders, reflecting the high threshold for correction of GAA deficiency in the skeletal muscle of Pompe disease patients.

[0021] Even in cross-reactive immunologic material (CRIM)-positive infants with Pompe disease, a lack of complete efficacy from ERT has been observed. Children with Pompe disease have residual motor developmental delays and respiratory insufficiency. Hypernasal speech and swallowing difficulties indicated residual oromotor abnormalities despite long-term ERT in infantile-onset Pompe disease. Many young children with Pompe disease require temporary or long-term assisted ventilation. Strabismus and ptosis have been observed frequently among children with Pompe disease, while receiving ERT. Each of these abnormalities demonstrates a lack of complete efficacy from ERT. Patients with late-onset Pompe disease have severe pulmonary insufficiency may progress to respiratory failure while receiving ERT. ERT stabilized but did not reverse muscle weakness and pulmonary involvement in adult patients with Pompe disease. Many individuals with late-onset Pompe disease have residual gait abnormalities despite adherence to ERT, indicating a relative lack of response of leg muscles.

[0022] Accordingly, there exists a continuing need for new therapies for lysosomal storage disorders, such as Pompe disease. The present disclosure is directed to the premise that Pompe disease can be more effectively treated by increasing the cation- independent mannose 6- phosphate receptor (CI-MPR) mediated uptake of rhGAA with simultaneous β2 agonist administration. Accordingly, provided herein are methods of treating lysosomal storage disorders comprising an adjuvant therapy comprising a β2 agonist to enhance efficacy of ERT.

[0023] It must also be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "β2 agonist" is a reference to one or more β2 agonists and equivalents thereof known to those skilled in the art, and so forth.

[0024] "Administering", when used in conjunction with a therapeutic, means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a subject, whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term "administering", when used in conjunction with a therapeutic, can include, but is not limited to, providing a therapeutic to a subject systemically by, for example, intravenous injection, whereby the therapeutic reaches the target tissue. Administering a composition or therapeutic may be accomplished by, for example, injection, oral administration, topical administration, or by these methods in combination with other known techniques. Administering may be self-administration, wherein the therapeutic or composition is administered by the subject themselves. Alternatively, administering may be administration to the subject by a health care provider.

[0025] "Providing", when used in conjunction with a therapeutic, means to administer a therapeutic directly into or onto a target tissue, or to administer a therapeutic to a subject whereby the therapeutic positively impacts the tissue to which it is targeted.

[0026] The terms, "treat" and "treatment," as used herein, refer to amelioration of one or more symptoms associated with the disease, prevention or delay of the onset of one or more symptoms of the disease, and/or lessening of the severity or frequency of one or more symptoms of the disease. In embodiments described herein, treatment can refer to improvement in mortality, improvement of cardiac status (e.g., increase of end-diastolic and/or end-systolic volumes, or reduction, amelioration or prevention of the progressive cardiomyopathy that is typically found in GSD-II) or of pulmonary function (e.g., increase in crying vital capacity over baseline capacity, and/or normalization of oxygen desaturation during crying, reduction in need for invasive or non-invasive ventilator support); improvement in neurodevelopment and/or motor skills (e.g., increase in AIMS score or 6MWT); reduction of glycogen levels in tissues of the individual affected by the disease; or any combination of these effects. In embodiments described here, treatment includes improvement of cardiac status, particularly in reduction or prevention of GSD-II-associated cardiomyopathy.

[0027] As used herein, the term "therapeutic" means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a subject. In part, embodiments described herein may be directed to the treatment of various lysosomal storage disorders, including, but not limited to, Pompe disease (GSD II), Gaucher disease, Fabry disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, or Niemann- Pick disease. In some embodiments, the lysosomal storage disorder is glycogen storage disease II (Pompe disease). In some embodiments, the glycogen storage disease II may be LOPD, juvenile-onset Pompe disease, or infantile Pompe disease.

[0028] The terms, "improve," "increase" or "reduce," as used herein, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A control individual is an individual afflicted with the same form of GSD-II (either infantile, juvenile or adult-onset) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).

[0029] The terms "therapeutically effective" or "effective", as used herein, may be used interchangeably and refer to an amount of a therapeutic composition or combination of therapeutic compositions described herein. For example, a therapeutically effective amount of a composition is an amount of the composition, and particularly the active ingredient, such as a beta-2 agonist and/or GAA, that generally achieves the desired effect. For example, the desired effect can be an improvement, prevention, or reduction of a particular disease state.

[0030] A "therapeutically effective amount" or "effective amount" of a composition is an amount necessary or sufficient to achieve the desired result or clinical outcome. For example, the desired result or clinical outcome can be an improvement, prevention, or reduction of a particular disease state. The therapeutic effect contemplated by the embodiments herein includes medically therapeutic, cosmetically therapeutic and/or prophylactic treatment, as appropriate. The specific dose of a compound administered according to embodiments of the present invention to obtain therapeutic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. However, the effective amount administered may be determined by the practitioner or manufacturer or patient in light of the relevant circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore, the above dosage ranges are not intended to limit the scope of the invention in any way. A therapeutically effective amount of the compound of embodiments herein is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in or on the tissue to achieve the desired therapeutic or clinical outcome.

[0031] As used herein, the term "consists of or "consisting of means that the composition or method includes only the elements, steps, or ingredients specifically recited in the particular claimed embodiment or claim.

[0032] As used herein, the term "consisting essentially of or "consists essentially of means that the composition or method includes only the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0033] Generally speaking, the term "tissue" refers to any aggregation of similarly specialized cells which are united in the performance of a particular function. [0034] The term "animal" as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals.

[0035] The term "patient" or "subject" as used herein is an animal, particularly a human, suffering from an unwanted disease or condition that may be treated by the therapeutic and/or compositions described herein.

[0036] The term "inhibiting" generally refers to prevention of the onset of the symptoms, alleviating the symptoms, or eliminating the disease, condition or disorder.

[0037] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0038] As used herein, "room temperature" means an indoor temperature of from about 20°C to about 25°C (68 to 77°F).

[0039] Throughout the specification of the application, various terms are used such as "primary," "secondary," "first," "second," and the like. These terms are words of convenience in order to distinguish between different elements, and such terms are not intended to be limiting as to how the different elements may be utilized.

[0040] By "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, as they refer to compositions, carriers, diluents, and reagents or other ingredients of the formulation, can be used interchangeably and represent that the materials are capable of being administered without the production of undesirable physiological effects such as rash, burning, irritation or other deleterious effects to such a degree as to be intolerable to the recipient thereof

[0041] Embodiments herein describe a novel therapeutic approach comprising administering a therapeutic agent as an adjunctive therapy for enzyme replacement therapy to treat a lysosomal storage disorder. It is believed that increasing CI-MPR expression could reduce the dosage requirements for ERT or a future gene therapy. Overall, it is believed that the availability of treatments that can improve efficacy of ERT for Pompe disease and other lysosomal storage disorders will reduce the costs of therapies for these diseases. β2 agonists are generally affordable and well-tolerated. This therapeutic strategy may also be applicable to other lysosomal storage disorders for which ERT is available, where therapy is complicated by inefficient receptor-mediated uptake of the therapeutic enzyme. Such lysosomal storage disorders include Gaucher disease, Fabry disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, or Niemann-Pick disease.

[0042] In some embodiments, a method of treating a lysosomal storage disorder in an individual comprises administering a lysosomal enzyme and another therapeutic agent. Certain embodiments are directed to the use of a lysosomal enzyme and another therapeutic agent in the manufacture of a medicament for the treatment of a lysosomal storage disorder. In some embodiments, the lysosomal storage disorder to be treated may be Pompe disease (GSD II), Gaucher disease, Fabry disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, or Niemann-Pick disease. In some embodiments, the lysosomal storage disorder is glycogen storage disease II. In some embodiments, the glycogen storage disease II may be late-onset Pompe disease (LOPD), juvenile-onset Pompe disease, or infantile Pompe disease. In some embodiments, the lysosomal enzyme and the other therapeutic agent are in a single composition. In some embodiments, the lysosomal enzyme and the other therapeutic agent may be in separate compositions. In such embodiments, the other therapeutic agent may be administered prior to, concurrently with, or after the lysosomal enzyme. In some embodiments, the lysosomal enzyme is acid a- glucosidase (GAA). In some embodiments, the therapeutic agent is a β2 agonist.

[0043] In some embodiments, the individual, patient, or subject being treated may be a human (fetus, child, adolescent, or adult human) having a lysosomal storage disorder, such as, for example, GSD-II (i.e., infantile GSD-II, juvenile GSD-II, or adult- onset GSD-II). In some embodiments, the individual may have residual GAA activity, or no measurable activity. In some embodiments, the individual having GSD-II may have GAA activity that is less than about 1% of normal GAA activity (infantile GSD-II), GAA activity that is about 1-10% of normal GAA activity (juvenile GSD-II), or GAA activity that is about 10-40% of normal GAA activity (adult GSD-II). In some embodiments, the individual may be CRIM-negative for endogenous GAA. In some embodiments, the individual is CRIM-positive for endogenous GAA. In some embodiments, the individual has been recently diagnosed with the disease. Early treatment (treatment commencing as soon as possible after diagnosis) may be important to minimize the effects of the disease and to maximize the benefits of treatment.

[0044] Without wishing to be bound, it is believed that adjunctive therapy, as described in the various embodiments herein, with a β2 agonist will enhance the response to ERT in Pompe disease by increasing the expression of CI-MPR. There is a deficiency of CI-MPR in type II myofibers, which were glycogen-laden despite ERT, of mice with Pompe disease. Applicants have demonstrated that treatment with a selective β2 agonist increased expression of CI-MPR in skeletal muscle, resulting in a marked improvement in the response to ERT in mice with Pompe disease.

[0045] In some embodiments, increasing expression of receptors for a lysosomal enzyme may be used in combination with enzyme replacement therapy, for example, administration and expression of a vector encoding the lysosomal enzyme in the individual. In some embodiments, useful vectors include viral vectors, such as an adeno-associated virus (AAV) vector. In some embodiments, the individual may receive enzyme replacement therapy prior to, concurrently with, or subsequent to, increasing expression of receptors for the lysosomal enzyme in the individual. In such combination therapies, the efficacy of the enzyme replacement therapy may be enhanced, including efficacy of the enzyme replacement in brain. In some embodiments, the activity of the lysosomal enzyme in the individual may be increased to a level greater than that observed in a patient receiving enzyme replacement therapy alone or in a patient prior to increasing expression of receptors for the lysosomal enzyme. In some embodiments, where increasing expression of receptors for the lysosomal enzyme is performed by administering a β2 agonist, the activity of the lysosomal enzyme in the individual is increased to a level greater than that observed in a patient receiving enzyme replacement therapy alone or receiving the β2 agonist alone.

[0046] Some embodiments herein are directed to a method for treating a lysosomal storage disorder (e.g. Pompe disease) by increasing a lysosomal enzyme receptor (e.g. CI-MPR) expression in target tissues. Accordingly, some embodiments of the present disclosure contemplate methods of treating a lysosomal storage disorder in an individual by administering, as an adjunctive therapy, a therapeutic agent to the individual, wherein the individual is undergoing or has undergone enzyme replacement therapy. [0047] In some embodiments, it is believed that increasing CI-MPR expression will enhance the uptake and lysosomal targeting of GAA and enhance biochemical correction in skeletal muscle, including in Pompe disease. Some embodiments are directed to methods of increasing CI-MPR expression comprising administering GAA and a β2 agonist. In some embodiments, a method of increasing CI-MPR expression in an individual having a lysosomal storage disorder comprises administering a β2 agonist as an adjuvant therapy, wherein the individual is undergoing or has undergone enzyme replacement therapy with acid alpha- glucosidase, a recombinant form thereof, or a functional equivalent thereof. In some embodiments, the lysosomal storage disorder is Pompe disease. Some embodiments herein describe a method of increasing receptor mediated uptake of a lysosomal enzyme, a recombinant form thereof, or a functional equivalent thereof in an individual having a lysosomal storage disorder, the method comprising administering a therapeutic agent as an adjuvant therapy to lysosomal enzyme replacement therapy.

[0048] In some embodiments of the methods of the invention, the lysosomal enzyme may be in a form that, when administered, targets tissues such as the tissues affected by the disease (e.g., heart, muscle). In embodiments, the lysosomal enzymes may include a human enzyme, recombinant enzyme, wild-type enzyme, synthetic enzyme, or a combination thereof. In embodiments, lysosomal enzymes may be selected from glucocerebrosidase (for the treatment of Gaucher disease; U.S. Pat. No. 5,879,680 and U.S. Pat. No. 5,236,838,) alpha-glucosidase (e.g., acid alpha-glucosidase) (for the treatment of Pompe disease; PCT International Publication No. WO 00/12740), alpha-galactosidase (e.g., alpha-gal, alpha-galactosidase or alpha-gal) (for the treatment of Fabry Disease; U.S. Pat. No. 5,401,650), alpha-n-acetylgalactosaminidase (for the treatment of Schindler Disease; U.S. Pat. No. 5,382,524), acid sphingomyelinase (for the treatment of Niemann- Pick disease; U.S. Pat. No. 5,686,240) and alpha-iduronidase for the treatment of Hurler, Scheie, or Hurler-Scheie disease (PCT International Publication No. WO 93/10244A1). In some embodiments, the lysosomal enzyme is selected from glucocerebrosidase, acid alpha- glucosidase, alpha-galactosidase, alpha-n-acetylgalactosaminidase, acid sphingomyelinase, alpha-iduronidase, or a combination thereof. In some embodiments, the lysosomal enzyme may be GAA. In some embodiments, the GAA may be human. In some embodiments, the human GAA is administered in its precursor form, as the precursor contains motifs which allow efficient receptor-mediated uptake of GAA. Alternatively, a mature form of human GAA that has been modified to contain motifs to allow efficient uptake of GAA, can be administered. In some embodiments, the GAA may be GAA, rhGAA, neo-rhGAA (modified recombinant human GAA with synthetic oligosaccharide ligands which is sold by Genzyme Corp.), reveglucosidase alpha (a fusion of IGF-2 and GAA sold by Biomarin Pharmaceuticals, Inc.), ATB200 (an rhGAA with a higher bis-M6P content) that may optionally be administered in combination with AT221 (an oral chaperone molecule -1- deoxynojirimycin) (sold by Amicus Therapeutics, Inc.; described in U.S. Pat. App. No. 12/616,670), or a combination thereof. In some embodiments, the rhGAA may be alglucosidase alfa (sold by Genzyme Corp. under the tradename Myozyme® (for infantile onset Pompe disease) and Lumizyme®).

[0049] In embodiments, the GAA may have a specific enzyme activity in the range of about 1.0-3.5 μιηοΐ/min/mg protein, preferably in the range of about 2-3.5 μιηοΐ/min/mg protein. In some embodiments, the GAA has a specific enzyme activity of at least about 1.0 μιηοΐ/min/mg protein; more preferably, a specific enzyme activity of at least about 2.0 μιηοΐ/min/mg protein; even more preferably, a specific enzyme activity of at least about 2.5 μιηοΐ/min/mg protein; and still more preferably, a specific enzyme activity of at least about 2.75 μιηοΐ/min/mg protein.

[0050] According to some embodiments, a method of treating a lysosomal storage disease may be by increasing expression of receptors for the lysosomal enzyme, or otherwise increasing cell surface density of such receptors, in an individual in need thereof. In some embodiments, the therapeutic agent may be selected from a growth hormone (e.g., human growth hormone), an autocrine glycoprotein (e.g., Follistatin), and a β2 agonist. Such therapeutic agents may selectively modulate expression of receptors for particular lysosomal enzymes. Expression of receptors for a lysosomal enzyme may also be increased by behaviors, such as exercise. In some embodiments, a β2 agonist may be administered to an individual suffering from adult-onset or late-onset glycogen storage disease II, or a patient who presents with only partial enzyme deficiency, wherein administering the β2 agonist results in biochemical correction of the enzyme deficiency in target tissues and improved motor function.

[0051] β2 agonists are molecules that stimulate the

receptor. Numerous β2 agonists are known in the art and may be used in the therapeutic methods of the invention. In some embodiments, the β2 agonist used in embodiments herein may be selected from albuterol, arbutamine, bambuterol, befunolol, bitolterol, bromoacetylalprenololmenthane, broxaterol, carbuterol, cimaterol, cirazoline, clenbuterol, clorprenaline, denopamine, dioxethedrine, dopexamine, ephedrine, epinephrine, etafedrine, ethylnorepinephrine, etilefrine, fenoterol, formotorol, hexoprenaline, higenamine, ibopamine, isoetharine, isoproterenol, isoxsuprine, mabuterol, metaproterenol, methoxyphenamine, norepinephrine, nylidrin, oxyfedrine, pirbuterol, prenalterol, procaterol, propranolol, protokylol, quinterenol, ractopamine, reproterol, rimiterol, ritodrine, salmefamol, soterenol, salmeterol, terbutaline, tretoquinol, tulobuterol, xamoterol, zilpaterol, zinterol, or a combination thereof. In some embodiments, β2 agonists used in the disclosed methods do not interact, or show substantially reduced interaction, with βΐ- adrenergic receptors. In some embodiments, the β2 agonist is a selective β2 agonist. In embodiments, the β2 agonist is clenbuterol, albuterol, formoterol, salmeterol, or a combination thereof. In embodiments, the β2 agonist is clenbuterol. In embodiments, the β2 agonist is albuterol.

[0052] Also encompassed by the instant disclosure are methods of increasing efficacy of a lysosomal storage disease therapy, e.g., substrate deprivations and small molecule therapies, lysosomal enzyme replacement therapy, including gene therapy (e.g., transfection of cells in a patient with a vector encoding a deficient lysosomal enzyme), or any other form of therapy where the levels of the deficient lysosomal enzyme in a patient are supplemented. For example, these therapies may comprise increasing expression of receptors for a lysosomal enzyme, for example, by administering a therapeutically effective amount of a therapeutic agent (e.g. a β2 agonist).

[0053] In some embodiments, the other therapeutic agent may be administered in combination with a second therapeutic agent or treatment. In some embodiments, the therapeutic agents or treatments may be administered concurrently or consecutively in either order. For concurrent administration, in some embodiments, the therapeutic agents may be formulated as a single composition or as separate compositions. The optimal method and order of administration of the therapeutic agents capable of increasing expression of a receptor for a lysosomal enzyme and a second therapeutic agent or treatment may be ascertained by those skilled in the art using conventional techniques and in view of the information set out herein.

[0054] The disclosed combination therapies may elicit a synergistic therapeutic effect, i.e., an effect greater than the sum of their individual effects or therapeutic outcomes. Measurable therapeutic outcomes are described herein. For example, a synergistic therapeutic effect may be an effect of at least about two-fold greater than the therapeutic effect elicited by a single agent, or the sum of the therapeutic effects elicited by the single agents of a given combination, or at least about five-fold greater, or at least about ten-fold greater, or at least about twenty-fold greater, or at least about fifty-fold greater, or at least about one hundred-fold greater. A synergistic therapeutic effect may also be observed as an increase in therapeutic effect of at least 10% compared to the therapeutic effect elicited by a single agent, or the sum of the therapeutic effects elicited by the single agents of a given combination, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or more. A synergistic effect may also be an effect that permits reduced dosing of therapeutic agents when they are used in combination.

[0055] In some embodiments, for the treatment of lysosomal storage disorders, a therapeutic agent of embodiments described herein may be administered to a patient in combination with a lysosomal enzyme. In some embodiments, the other therapeutic agent and lysosomal enzyme may be components of a single pharmaceutical composition. In some embodiments, the other therapeutic agent and lysosomal enzyme may be components of separate pharmaceutical compositions that are mixed together before administration. In some embodiments, the other therapeutic agent and lysosomal enzyme may be components of separate pharmaceutical compositions that are administered separately. In some embodiments, the other therapeutic agent and the lysosomal enzyme may be administered simultaneously, without mixing (e.g., by delivery of the β2 agonist on an intravenous line by which the lysosomal enzyme is also administered). In some embodiments, the other therapeutic agent may be administered separately (e.g., not admixed), but within a short time frame (e.g., within 24 hours) prior to or subsequent to administration of the lysosomal enzyme. A synergistic effect may support reduced dosing of ERT when used with the other therapeutic agent and a reduced dosing of the other therapeutic agent. [0056] In embodiments, a lysosomal enzyme, such as GAA, may be administered in a form that targets tissues such as the tissues affected by the disease (e.g., heart, muscle, brain). The lysosomal enzyme may be optionally administered in conjunction with other agents, such as antihistamines or immunosuppressants or other immunotherapeutic agents that counteract anti-lysosomal enzyme antibodies. For gene therapy, genes encoding the aforesaid lysosomal enzymes are described in the preceding patent publications as well. The patents and published patent applications mentioned in this paragraph are specifically incorporated herein by reference in their entirety, and in particular, the disclosures contained therein with respect to the indicated enzymes, and sequences encoding such enzymes, are also incorporated by reference.

[0057] In some embodiments, administration of a lysosomal enzyme may also encompass administration of a functional equivalent of a lysosomal enzyme. A functional equivalent may include a compound different from the lysosomal enzyme that, when administered to the patient, replaces the function of the lysosomal enzyme to treat the lysosomal storage disorder. Such functional equivalents may include mutants, analogs, and derivatives of lysosomal enzymes.

[0058] In some embodiments, for the treatment of Pompe disease, the relevant lysosomal enzyme is an acid alpha-glucosidase. In some embodiments, the acid alpha- glucosidase may be a precursor form of human acid alpha-glucosidase, such as recombinant human acid alpha-glucosidase produced in Chinese hamster ovary (CHO) cell cultures. As another example, for the treatment of Gaucher disease, the relevant lysosomal enzyme is glucocerebrosidase, modified glucocerebrosidase or CEREZYME® enzyme.

[0059] In some embodiments, a method of treating a patient having GSD II comprises administering a β2 agonist as an adjunctive therapy for enzyme replacement therapy with acid alpha-glucosidase. In a related aspect of the invention is provided a method of treating a patient having Pompe disease characterized by reduced or deficient activity of acid a-glucosidase by (a) administering acid a-glucosidase replacement therapy to the patient; and (b) administering a β2 agonist to the patient. In accordance with the disclosed methods, it is believed that levels of Cation Independent Mannose-6-Phosphare Receptors (CI-MPR) are increased in the patient, activity of acid α-glucosidase is increased in the patient, and/or levels of glycogen are decreased in the patient. In addition, the amount of acid alpha-glucosidase required in the ERT may be reduced and the biochemical correction in muscle biopsies may be increased from baseline.

[0060] In the embodiments described herein, a therapeutically effective amount of the other therapeutic agent is administered. In some embodiments, the therapeutically effective amount of clenbuterol is about 40 μg/day to about 160 μg/day. In some embodiments, the therapeutically effective amount is about 20 μg/day to about 2100 μg/day, about 20 μg/day to about 720 μg/day, about 20 μg/day to about 500 μg/day, about 20 μg/day to about 300 μg/day, about 20 μg/day to about 200 μg/day, about 40 μg/day to about 2100 μg/day, about 40 μg/day to about 720 μg/day, about 40 μg/day to about 500 μg/day, about 40 μg/day to about 300 μg/day, about 40 μg/day to about 200 μg/day, about 80 μg/day to about 2100 μg/day, about 80 μg/day to about 720 μg/day, about 80 μg/day to about 500 μg/day, about 80 μg/day to about 300 μg/day, about 80 μmg/day to about 200 μg/day, or a range between any two of these values. In some embodiments, the therapeutically effective amount of albuterol is about 4 mg/day to about 16 mg/day. In some embodiments, the therapeutically effective amount is about 2 mg/day to about 20 mg/day, about 2 mg/day to about 16 mg/day, about 2 mg/day to about 10 mg/day, about 2 mg/day to about 5 mg/day, about 4 mg/day to about 20 mg/day, about 4 mg/day to about 16 mg/day, about 4 mg/day to about 10 mg/day, about 4 mg/day to about 5 mg/day, about 8 mg/day to about 20 mg/day, about 8 mg/day to about 16 μg/day, about 8 mg/day to about 10 μg/day, or a range between any two of these values. In embodiments, the therapeutically effective amount for a particular individual may be varied (e.g., increased or decreased) over time, depending on the needs of the individual.

[0061] In some embodiments, the therapeutic agents may be administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or a range between any two of these values. In some embodiments, the therapeutic agents may be administered at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks, or a range between any two of these values. In some embodiments, the therapeutic agents may be administered using single or divided doses of every 60, 48, 36, 24, 12, 8, 6, 4, or 2 hours, or a range between any two of these values, or a combination thereof. [0062] The methods of the present disclosure contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time. In embodiments, the therapeutic agent may be administered at regular intervals (i.e., periodically) and on an ongoing basis, depending on the nature and extent of effects of the lysosomal storage disease, and also depending on the outcomes of the treatment. In some embodiments, the therapeutic agents' periodic administrations may be bimonthly, monthly, biweekly, weekly, twice weekly, daily, twice a day, three times a day, or more often a day. Administrative intervals may also be varied, depending on the needs of the patient. For example, in some embodiments, in times of physical illness or stress, if anti-lysosomal enzyme antibodies become present or increase, or if disease symptoms worsen, the interval between doses may be decreased. Therapeutic regimens may also take into account half-life of the administered therapeutic agents of embodiments herein.

[0063] In the embodiments described herein, a therapeutically effective amount of a lysosomal enzyme is administered. In some embodiments, the lysosomal enzyme is administered as part of a lysosomal enzyme replacement therapy. In some embodiments, the therapeutically effective amount of the lysosomal enzyme (e.g. GAA) is about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 5 mg/kg to about 50 mg/kg, about 5 mg/kg to about 40 mg/kg, about 5 mg/kg to about 30 mg/kg, about 5 mg/kg to about 20 mg/kg, about 10 mg/kg to about 50 mg/kg, about 10 mg/kg to about 40 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, less than about 50 mg/kg, less than about 40 mg/kg, less than about 30 mg/kg, less than about 25 mg/kg, less than about 20 mg/kg, less than about 15 mg/kg, less than about 10 mg/kg, less than about 5 mg/kg, or a range between any two of these values. In some embodiments, the effective dose for a particular individual may be varied (e.g., increased or decreased) over time, depending on the needs of the individual. For example, in times of physical illness or stress, or if anti-enzyme antibodies become present or increase, or if disease symptoms worsen, the amount may be increased.

[0064] In embodiments, the therapeutically effective amount of the lysosomal enzyme (or composition or medicament containing the lysosomal enzyme) may be administered at regular intervals, depending on the nature and extent of the disease's effects, and on an ongoing basis. Administration at a "regular interval," as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In some embodiments, the therapeutic agent's periodic administrations may be bimonthly, monthly, biweekly, weekly, twice weekly, daily, twice a day, three times a day, or more often a day. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual. For example, in times of physical illness or stress, if anti-enzyme antibodies become present or increase, or if disease symptoms worsen, the interval between doses may be decreased. In some embodiments, a therapeutically effective amount of 10 mg GAA/kg body weight may be administered weekly. In some embodiments, a therapeutically effective amount of 5 mg GAA/kg body weight may administered twice weekly.

[0065] In some embodiments, the lysosomal enzyme may be administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or a range between any two of these values. In some embodiments, the lysosomal enzyme may be administered at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks, or a range between any two of these values. In some embodiments, the lysosomal enzyme may be administered using single or divided doses of every 60, 48, 36, 24, 12, 8, 6, 4, or 2 hours, or a range between any two of these values, or a combination thereof. For example, in some embodiments, for the treatment of Pompe disease, the lysosomal enzyme, functional equivalent thereof, or gene may be administered once every about one to about two, about two to about three, about three to about four, or about four to about five weeks.

[0066] In some embodiments, the other therapeutic agent may be administered prior to, or concurrently with, or shortly thereafter, the lysosomal enzyme, functional equivalent thereof or gene encoding such enzyme. In some embodiments, the other therapeutic agent may be administered sufficiently prior to administration of the lysosomal enzyme so as to permit modulation (e.g., up-regulation) of the target cell surface receptors to occur, for example, at least about two to about three days, about three to about four days, or about four to about five days before the lysosomal enzyme is administered. For example, in some embodiments, in the case of Pompe disease, a β2 agonist may be administered to a patient about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, or 1, 2, 3, 4, 5, 6, 7, 8 days, prior to administration of acid alpha- glucosidase enzyme, modified acid alpha- glucosidase or a functional equivalent thereof.

[0067] As known by those of skill in the art, the optimal dosage of therapeutic agents useful in the invention depend on the age, weight, general health, gender, and severity of the lysosomal storage disease of the individual being treated, as well as route of administration and formulation. A skilled practitioner is able to determine the optimal dose for a particular individual. Additionally, in vitro or in vivo assays may be employed to help to identify optimal dosage ranges, for example, by extrapolation from dose-response curves derived from in vitro or animal model test systems.

[0068] The therapeutic agents of embodiments herein may be administered by any suitable route, including administration by inhalation or insufflation (either through the mouth or the nose) or oral, sublingual, buccal, parenteral, topical, subcutaneous, intraperitoneal, intravenous, intrapleural, intraocular, intraarterial, rectal administration, or within/on implants, e.g., matrices such as collagen fibers or protein polymers, via cell bombardment, in osmotic pumps, grafts comprising appropriately transformed cells, etc.

[0069] Some embodiments are directed to a pharmaceutical composition comprising the other therapeutic agent and a pharmaceutically acceptable carrier or excipient. Some embodiments are directed to a pharmaceutical composition comprising the lysosomal enzyme and a pharmaceutically acceptable carrier or excipient. Some embodiments are directed to a pharmaceutical composition comprising the other therapeutic agent and a lysosomal enzyme. In some embodiments, a pharmaceutical composition may comprise GAA and a β2 agonist. The compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.

[0070] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally-acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. Formulation also varies according to the route of administration selected (e.g., solution, emulsion, capsule).

[0071] Pharmaceutically acceptable carriers can include inert ingredients which do not interact with the β2 agonist, lysosomal enzyme and/or other additional therapeutic agents. These carriers include sterile water, salt solutions (e.g., NaCl), physiological saline, bacteriostatic saline (saline containing about 0.9% benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer' s-lactate saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, dextrose, lactose, trehalose, maltose or galactose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose and polyvinyl pyrolidone, as well as combinations thereof. The compositions may be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, pH buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. In addition, the compositions of the invention may be lyophilized (and then rehydrated) in the presence of such excipients prior to use.

[0072] Standard pharmaceutical formulation techniques as known in the art can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Methods for encapsulating compositions. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose or magnesium carbonate. For example, a composition for intravenous administration typically is a solution in a water- soluble carrier, e.g., sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0073] In some embodiments, the therapeutic agents may be administered as a neutral compound or as a salt or ester. Pharmaceutically acceptable salts may include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic or tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol histidine, and procaine. For instance, salts of compounds containing an amine or other basic group can be obtained by reacting with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base such as a hydroxide base. Salts of acidic functional groups contain a countercation such as sodium or potassium.

[0074] While embodiments set forth herein are described in terms of "comprising", all of the foregoing embodiments also include compositions and methods that consist of only the ingredients or steps recited or consist essentially of the ingredients and steps recited, and optionally additional ingredients or steps that do not materially affect the basic and novel properties of the composition or method.

[0075] This disclosure and embodiments illustrating the method and materials used may be further understood by reference to the following non-limiting examples.

EXAMPLE 1: Phase I/II study of oral clenbuterol in adult subjects with Pompe disease

[0076] The safety and efficacy of a β2 agonist as adjunctive therapy in subjects with LOPD on ERT will be determined. This study will undertake the systematic evaluation of β2 agonist treatment as an adjunctive therapy to ERT in 20 adult subjects. Individuals with LOPD have muscle weakness without cardiac involvement, which will increase safety and the likelihood of demonstrating efficacy. Endpoints will include the 6MWT, muscle function testing, and pulmonary function testing.

[0077] This is a double-blinded, placebo-controlled Phase I/II study of oral clenbuterol in adult subjects with Pompe disease, all of whom will have been compliant with ERT at a stable dose for > 12 months. Patients enrolled in the pilot study of albuterol will stop taking that drug for > 12 weeks before enrolling in the clenbuterol protocol. A muscle biopsy will be performed at Baseline and at Week 52 (Table 2). This study includes baseline evaluations at the Week 0 and Week 6 visits to assess the rate of change in muscle function, prior to the 3 :2 randomization to oral clenbuterol or placebo at Week 6. Clenbuterol will be over-encapsulated to allow for placebo administration. The placebo will consist of the same capsules used to disguise clenbuterol tablets, although placebo capsules will contain only dextrose. There will be two phone visits with the subjects to evaluate AEs, one week following the initial administration of clenbuterol at Week 7, and one week after the dose increase at Week 13. Blood sampling for safety testing will be performed at Week 0, 12, 18, and 52 visits.

Table 2: Testing Schedule

Muscle biopsy X X

[0078] The initial dose of clenbuterol will be 40 meg (one capsule containing two 20 meg tablets) each morning for one week, and the Week 7 phone visit will inquire about any AEs from daily low-dose clenbuterol. The dose of clenbuterol may be reduced for subjects who experience significant side effects to lessen their symptoms as reported for other studies involving β2 agonists, and allow those subjects to continue in the study. If the single dose is well tolerated, the clenbuterol dose will be increased to 40 meg (one capsule) BID for the next 5 weeks until the week 12 visit. If the 40 meg BID per oral has been well tolerated at the Week 12 visit, the dose will be increased to 80 meg (2 capsules) each morning and 40 meg (one capsule) each evening for one week. The Week 13 phone visit will inquire about any AEs, and if the 80 meg morning dose has been well tolerated, the subjects will increase to 80 meg (2 capsules) BID until the week 18 visit. Subjects randomized to placebo will go through the same dose escalations and monitoring, albeit while taking capsules filled with dextrose. This strategy for advancing dosages has been effective in a study with albuterol in LOPD (see Fig. 4).

[0079] At Baseline (Day 0) each subject will provide written consent and undergo a screening evaluation to determine eligibility. Screening will consist of taking the medical history, a physical examination, and an electrocardiogram (EKG). Baseline pulmonary function testing will be obtained, and urine will be collected for biomarker analysis. Exclusion criteria will include abnormal EKG, hyperthyroidism, taking incompatible medications, pregnancy, or hypertension. Muscle function and strength testing will be performed on Day 0, and muscle biopsy will be performed on Day 1. The subject will return after 6 weeks for repeat muscle function and strength testing, prior to randomization. The Week 12 visit will assess safety and tolerability, and reassess muscle function testing. The Week 18 visit will evaluate safety and efficacy by performing blood testing, EKG, PFT, and muscle strength and function testing. At Week 52 safety and efficacy will be evaluated prior to a repeat muscle biopsy to determine the effect of clenbuterol on CI-MPR and glycogen content.

[0080] It is expected that oral clenbuterol will be well-tolerated in subjects with LOPD. The strategy for advancing dosages of clenbuterol gradually will reduce adverse effects and prevent attrition.

EXAMPLE 2: Effect of Clenbuterol upon receptor-mediated uptake of rhGAA

[0081] The effect of β2 agonist therapy upon receptor-mediated uptake of rhGAA in subjects with LOPD will be determined. The effects of β2 agonist therapy upon CI-MPR expression will be evaluated in adult subjects with LOPD undergoing ERT with rhGAA during a 12 week study. The dose will be increased after 6 weeks to optimize its effect, if the initial dose is well tolerated. The impact of enhanced CI-MPR-mediated uptake of GAA will be analyzed by comparing muscle function and biochemical correction of glycogen accumulation in muscle at baseline and during clenbuterol administration. The urinary biomarker, Glc₄, will be monitored. These studies will reveal any correlation between biochemical correction and clinical endpoints.

[0082] The impact of enhanced CI-MPR-mediated uptake of GAA will be analyzed by comparing muscle function, pulmonary function, and biochemical correction of muscle in subjects with LOPD treated with ERT, both prior to and during simultaneous β2 agonist therapy. Clenbuterol therapy will be combined with ERT to evaluate benefits of increased CI-MPR expression upon efficacy in adult subjects with Pompe disease during a

52 week study. Subjects will return for safety and efficacy monitoring after 6, 12, and 46 weeks of clenbuterol therapy. The efficacy of clenbuterol treatment during ERT in subjects with LOPD will be evaluated with muscle and pulmonary function testing as the primary endpoints. A secondary endpoint, the urinary Glc₄ biomarker, will be monitored when the subjects are evaluated at baseline, Week 6, Week 12, and Week 52. The impact of enhanced CI-MPR-mediated uptake of GAA will be analyzed by comparing the muscle function, pulmonary function, and biochemical correction of muscle in subjects with LOPD treated with ERT, both prior to and during simultaneous β2 agonist therapy. [0083] Muscle strength and muscle function will be evaluated with measures of impairment, function, participation, and quality of life at Week 0, 6 12, 18 and 52. Isolated muscle strength will be tested with hand held dynamometry as performed previously (Fig. 3A). Functional strength measures will include classic upper and lower extremity functional grades, GMFM, timed functional tests, and graded functional tests which will be included to allow scoring with the Gait, Stairs, Gowers, Chair (GSCS), and the Quick Motor Function Test, all of which have been used with LOPD, and which offer quantitative assessment of activities of elevation against gravity and gait. Endurance will be assessed with the 6MWT. Pulmonary function will be assessed in both the supine and upright positions to increase sensitivity for abnormalities detected in Pompe disease. Fatigue will be assessed with the Fatigue Severity Scale, level of disability will be assessed with the Rotterdam Handicap Scale, and quality of life will be measured with a SF36 health survey .

[0084] Pulmonary function testing will include measuring FVC, maximum voluntary ventilation (MVV), and will be measured by electronic spirometer. Pulmonary function will be assessed in both the supine and upright positions to increase sensitivity for abnormalities detected in Pompe disease.

[0085] Subjects will undergo a muscle biopsy of the quadriceps at the baseline and final visits in this study. The muscle biopsy will be performed in the EMG Clinic at Duke University Medical Center. The needle muscle biopsy is performed under local anesthesia, and subjects have not experienced adverse effects following this procedure beyond transient local pain following the procedure. Other risks associated with the procedure include hematoma, infection and scar formation. The muscle biopsy will be evaluated for biochemical correction as demonstrated by GAA activity and glycogen content, and for CI-MPR expression by Western blot analysis.

[0086] Oral clenbuterol treatment should increase the performance on muscle strength, muscle functional testing, and pulmonary function testing of subjects with LOPD, based upon published data regarding the outcomes of clinical trials of ERT in subjects with LOPD. The effects of clenbuterol with ERT might be dramatic enough to produce significant improvements in muscle strength and function. Furthermore, increased biochemical correction in the muscle biopsies should be obtained following clenbuterol treatment, in comparison with the baseline muscle biopsy (Fig. IB). [0087] Clenbuterol, a β2 agonist drug similar to albuterol, enhanced CI-MPR expression and increased efficacy from ERT (Fig. 1), thereby confirming the key role of CI- MPR with regard to replacement therapy in Pompe disease. To compare the effects of albuterol and clenbuterol, groups of three month-old tolerant GAA-KO mice were treated with four weekly doses of rhGAA (20 mg/kg body weight), with or without concurrent β2- agonist treatment. Tolerant GAA mice do not form anti-GAA antibodies or develop hypersensitivity reactions during ERT with rhGAA, similar to the majority of patients with LOPD. The greater efficacy of clenbuterol treatment, in comparison with albuterol treatment, was demonstrated by enhanced efficacy in comparison with ERT alone as follows: 1) Rotarod latency was increased reflecting improved neuromuscular function; 2) glycogen content was significantly decreased in the diaphragm, quadriceps, gastrocnemius, EDL, and soleus muscles; and 3) clenbuterol treatment alone did not achieve efficacy with regard to muscle testing or performance in GAA-KO mice. Subsequently, urinary Glc₄ was analyzed to evaluate it as a biomarker for the evaluation of adjunctive clenbuterol therapy. Urinary Glc₄ was significantly reduced by adjunctive therapy with clenbuterol (Fig. 2), further validating this noninvasive biomarker for monitoring the response to therapy in Pompe disease.

[0088] 16 subjects were previously enrolled with LOPD in a natural history study that evaluated muscle and respiratory involvement. That study was interrupted by the initiation of the clinical trial of ERT in LOPD, which recruited the majority of our subjects and prevented studying the progression of untreated LOPD. However, abnormalities of muscle and pulmonary function testing (Fig. 3) were demonstrated. Dynamometry revealed significant muscle weakness in a pattern typical for LOPD in 16 subjects, which primarily involved proximal leg muscles (Fig. 3A). Pulmonary function testing was abnormal in LOPD subjects, in comparison with established normal values (Fig. 3B). In a study of the long-term response to ERT on a LOPD patient with very advanced muscle involvement, improvement was shown in timed functional tests, the Gross Motor Function Measure (GMFM), and pulmonary function testing. These tests will be performed during the proposed Phase 1, Phase I/II trial to reveal efficacy from adjunctive therapy with clenbuterol in LOPD. EXAMPLE 3: Albuterol and Low-Dose Clenbuterol as Adjunctive Therapy for ERT

[0089] Clenbuterol treatment has been shown to enhance the biochemical correction of muscle from ERT in mice with Pompe disease. Therefore, the efficacy of adjunctive p2-agonists was evaluated. Dose-related side effects have been observed in association with p2-agonist treatment, and therefore clenbuterol was administered at a lower dose. A second p2-agonist, albuterol, was evaluated at a high dose. Low dose clenbuterol reduced glycogen content in all striated muscles evaluated with the exception of tibialis anterior, in comparison with ERT alone. Albuterol was less effective than clenbuterol. Both p2-agonists increased Rotarod latency, demonstrating improved neuromuscular function.

[0090] The effect of P2-agonist treatment was further evaluated by quantifying CI-MPR in tissues. CI-MPR detection by Western blot analysis revealed that clenbuterol treatment increased CI-MPR in the EDL muscle and brain. Histopathology was performed to examine brain involvement, and glycogen accumulations were detected in the cerebellum with ERT alone. Both albuterol and clenbuterol reduced the glycogen staining in the cerebellum and hippocampus, in comparison with ERT alone. Increased CI-MPR expression correlated with increased biochemical correction in muscle and brain, and with improved muscle function following p2-agonist treatment. The effect of clenbuterol was further demonstrated to enhance efficacy and reverse neuromuscular involvement from low- dose gene therapy in GAA-KO mice.

[0091] The mechanism of P2-agonist treatment was further evaluated in GAA- KO mice with muscle-specific knockout of CI-MPR ("double-knockout" or DKO mice). DKO mice should have a decreased response to the combination of p2-agonist treatment, if CI-MPR modulates the effect of p2-agonists upon ERT. Glycogen content of the heart, diaphragm, and gastrocnemius was only very slightly reduced following high dose clenbuterol and ERT, in comparison with mock treatment. Clenbuterol by itself failed to decrease glycogen content in GAA-KO mice that did express CI-MPR, confirming that biochemical correction depended on ERT, not the P2-agonist drug by itself. In order to estimate the relative contribution of CI-MPR to the effect of clenbuterol, the reduction in glycogen content achieved by combination therapy (ERT + clenbuterol) in GAA-KO and DKO mice was calculated. A greater degree of glycogen clearance was demonstrated in GAA-KO mice, in comparison with DKO mice. Therefore, the effect of clenbuterol was shown to be dependent upon CI-MPR expression.

[0092] Validation of the urinary biomarker for LOPD: The urinary biomarker for Pompe disease, urinary glucose tetrasaccharide (Glc₄), has been validated in the randomized trial of ERT in subjects with LOPD. Urinary Glc₄ was monitored in 60 subjects in the Myozyme treated group and 30 subjects in the placebo group for up to 78 weeks. The difference in mean Glc₄ values at baseline and 12 weeks were compared for each group. The mean Glc₄ value for the treated cohort showed a significant decrease after 12 weeks (p = 0.0002), whereas no change was observed for the placebo group (Fig. 5). These data validated the urinary biomarker as an endpoint for the evaluation of ERT in LOPD, including ERT in combination with adjunctive therapy with clenbuterol.

EXAMPLE 4: Albuterol as Adjunctive Therapy in Patients with LOPD

[0093] A study of albuterol as adjunctive therapy in patients with LOPD on ERT has been initiated. The design is similar to the above example, although without a placebo group (Fig. 4A). Briefly, subjects undergo a baseline evaluation including blood safety testing, EKG, pulmonary function testing, muscle function and strength testing, and muscle biopsy at Week 0, and subjects return at Week 6 and 12 for follow-up evaluations. At Week 6 the dose of albuterol is increased, barring dose-limiting AEs. Repeat EKG, pulmonary function testing, and muscle biopsy are completed at Week 12. Only subjects stably treated with ERT for greater than two years have been enrolled in this study, and inclusion/exclusion criteria are identical to those for the proposed study of clenbuterol. Seven subjects have had the initial muscle biopsy, 5 have returned for the Week 6 visit, and two have completed the Week 12 visit including a second muscle biopsy. The 6MWT distance at the indicated study visits is shown in Fig. 4B. In addition, the right and left hand grip strength tested by dynamometry is shown in Figs. 4C and 4B.

[0094] Preliminary data revealed that the 6MWT increased in all 5 subjects at the Week 6 visit following initiation of albuterol at baseline. Two of 7 subjects have completed only the Week 0 visit, having been enrolled < 6 weeks (data not shown). An increased 6MWT distance from baseline to week 6 in the 6MWT for all 5 subjects was observed. The change in 6MWT on ERT was assessed retrospectively for subjects followed at Duke University. Prior 6MWT data was retrieved for 3 of 5 subjects, which allowed calculation of change in 6MWT between 3-6 months earlier and Week 0 (Pre-albuterol). The 6MWT had decreased (Subject 4), stayed the same (Subject 1 ), or increased slightly (Subject 5) prior to study entry (Prealbuterol); whereas the 6MWT increased for all 3 subjects from baseline to Week 6 (Albuterol). This group of subjects had a negative mean change in 6MWT (-13 +/- 30 meters) in the months prior to stalling albuterol (Prealbuterol), which was reversed after 6 weeks of albuterol (32 +/- 19 meters for those 3 subjects). When the available retrospective data was compared with data for all subjects following 6 weeks of albuterol therapy, the increase in 6MWT was statistically significant (Pre-albuterol versus Albuterol; p = 0.03).

[0095] The mean change in 6MWT for the initial group of 5 subjects over the first 6 weeks was 30 +/- 14 meters, which is equivalent to the increased time in the 6MWT observed after 24 weeks in the initial study of ERT in LOPD. Moreover, the change in 6MWT after 12 weeks in the study of van der Ploeg et al. was only 14 +/- 3 meters, less than 50% of the observed change after 6 weeks in this pilot study of adjunctive albuterol. Thus, an early increase in 6MWT at Week 6 was observed in a stable population of subjects with LOPD on ERT and albuterol, which has exceeded the increase observed at early timepoints in the initial trial of ERT. Finally, anecdotal reports indicated benefits that were associated with improved responses in muscle function testing: 1) a woman could now stand up from sitting the floor much more easily (time for supine to standing decreased from 30 seconds to 11 seconds); and 2) a man can now readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). These preliminary data suggest that adjunctive therapy with β2 agonists will be efficacious in LOPD.

[0096] This study had several limitations that will be addressed in future studies, including: 1) lack of placebo controls, 2) performing only a single evaluation at baseline, which prevented calculating the baseline change in 6MWT prior to initiating study drug, and 3) monitoring for only 12 weeks to detect efficacy.

Safety demonstrated for up to 12 weeks

[0097] Subjects in the study of albuterol have been monitored for safety at Week

6 and Week 12 visits, and with phone visits at Week 1 and Week 7. The albuterol dose was gradually increased to minimize AEs, starting with 4 mg each morning per oral for the first week. If no AEs greater than mild were reported at the Week I phone visit, the dose was increased to 4 mg BID per oral. If the albuterol dose was similarly well-tolerated at the Week 6 visit, the dose was increased to 8 mg in the morning, and remained at 4 mg in the evening for the next week. Finally, if the Week 7 phone visit revealed no more than mild AEs, the evening dose was increased to 8 mg. This dose titration has prevented attrition related to the effects of albuterol, and all 5 subjects who completed the Week 6 visit have tolerated the 8 mg BID dose. Only mild AEs have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding).

EXAMPLE 5: Alternative β2 Agonists as Adjunctive Therapy for Pompe disease

[0098] This study evaluated 4 new drugs in GAA-KO mice in combination with an adeno-associated virus (AAV) vector encoding human GAA. The dosage for each drug was selected to induce muscle hypertrophy with an associated increased expression of CI- MPR, analogous to clenbuterol's effects. Three alternative β2 agonists and dehydroepiandrosterone (DHEA) were tested, given that these drugs were expected to upregulate both Igf-1 and downstream Igf-2R/CI-MPR, similar to clenbuterol. See Table 3.

Table 3: Small molecule thera ies evaluated in combination with gene therapy

*Administered in drinking water. Results

[0099] Mice were injected with AAV2/9-CBhGAApA [lE+11 vector particles (vp)] at a dose previously found to be partially effective at clearing glycogen storage from the heart following the induction of immune tolerance to GAA. Drugs were dosed continuously at dosages determined from the literature (Table 3). After 18 weeks striated muscles were analyzed for GAA and glycogen content. Heart GAA activity was significantly increased by either salmeterol (p<0.01) or DHEA (p<0.05), in comparison with untreated GAA-KO mice (Fig. 6A). Furthermore, glycogen content was reduced by treatment with DHEA (p<0.001), salmeterol (p<0.05), formoterol (p<0.01), or clenbuterol (p<0.01) in combination with the AAV vector, in comparison with untreated mice (Fig. 6A). The reduction of glycogen content in absence of significantly increased GAA activity has been observed following the addition of an adjunctive β2 agonist. Of note, glycogen content of the heart and skeletal muscle remained highly elevated in comparison with nearly undetectable amounts of glycogen observed in wildtype mice. The GAA activity and glycogen content of the diaphragm and quadriceps were not affected by any of the treatments (Fig. 6B), consistent data showing that heart muscle is more responsive to GAA replacement than skeletal muscle.

[0100] Functional testing was performed subsequently, and the wirehang test at 18 weeks following vector administration revealed that the combination of salmeterol and the AAV vector significantly increased latency in comparison with the AAV vector alone (p<0.001). Similarly, salmeterol with the vector increased latency significantly more than either DHEA (pO.001), formoterol (p<0.05), fenoterol (p<0.05), or clenbuterol (p<0.05) with the vector (Fig. 7A). No significant difference in body weight was observed between any of the treatments (Fig. 7B).

[0101] An important consideration with regard to adjunctive therapy is whether any effects are due to the adjuvant rather than the combined treatment. The most effective individual drugs, salmeterol and DHEA, were evaluated by themselves. No significant effect upon GAA activity of heart, glycogen content of heart, or wirehang latency was observed, in comparison with untreated mice (Fig. 8).

[0102] Further evaluation of CI-MPR and LC3 was performed by Western blotting (Fig. 9). Despite evidence that CI-MPR increased in skeletal muscle following β2 agonist administration, statistically significant increases were not observed in heart or quadriceps following administration of the four β2 agonists in this study (Fig. 9A-9B). However, the abnormally increased LC3-II previously described in the muscle of GAA-KO mice was significantly reduced in heart by administration of each of the four β2 agonists (Fig. 9B). Furthermore, administration of propranolol, a beta-blocker that increased the uptake of GAA but reduced efficacy from ERT, failed to reduce LC3-II (Fig. 9A,C). Reductions in LC3-II were consistent with reversal of abnormally accumulated autophagosomes previously described in GAA-KO mice. Discussion

[0103] Three alternative β2 agonists and dehydroepiandrosterone (DHEA) were evaluated in combination with gene therapy in GAA-KO mice. These drugs shared the ability to promote muscle hypertrophy, are available in the US, and were well-tolerated in rodent experiments. Mice were injected with AAV2/9-CBhGAA at a dose previously found to be partially effective at clearing glycogen storage from the heart. Heart GAA activity was significantly increased by either salmeterol or DHEA, in comparison with untreated mice. Furthermore, glycogen content was reduced by treatment with DHEA, salmeterol, formoterol, or clenbuterol in combination with the AAV vector. Functional testing with the wirehang test revealed that the combination of salmeterol and the AAV vector significantly increased latency in comparison with the other treatments. An important consideration with regard to adjunctive therapy is whether any effects are due to the adjuvant rather than the combined treatment. The most effective individual drugs were evaluated by themselves, and no significant effect was observed.

[0104] These four new agents were chosen for evaluation with gene therapy in GAA-KO mice, based upon prior evidence that β2 agonists inducing muscle hypertrophy were beneficial during GAA replacement in Pompe disease. As a positive control, mice were transgenic for a liver-specific human GAA transgene to induce immune tolerance to introduced GAA and were treated with clenbuterol at a low dose demonstrated to improve the response to ERT as described. Three new β2 agonists were chosen to be longer acting than albuterol, because the long-acting β2 agonist clenbuterol has been more efficacious than albuterol in rodent experiments. The dose-response for fenoterol and salmeterol has been equivalent to that for clenbuterol in previous rodent studies, and therefore these drugs were dosed similarly to clenbuterol to achieve muscle hypertrophy (Table 3). Moreover, these three β2 agonists have marketing approval from FDA and are available clinically, unlike clenbuterol. Finally, DHEA was chosen for: 1) its ability to promote muscle hypertrophy similarly to the β2 agonists, 2) availability in the US, and 3) lack of toxicity in rodent experiments.

[0105] Somewhat unexpectedly, performance on the wirehang test correlated with biochemical correction of the heart alone and not of skeletal muscles. However, this phenomenon has been observed following administration of ERT with propranolol. The observed improvement in muscle function might be attributable to greater cardiac function following biochemical correction of the heart, although cardiac function was not directly evaluated in either of these studies.

[0106] Limitations of the current experiment include a lack of detectable effect upon CI-MPR expression from adjunctive therapy, and a lack of effect of therapy upon the skeletal muscles. One limitation of the current experiment is that CI-MPR was not significantly increased by β2 agonist administration, in contrast to prior studies. This may reflect the variability of the individual response to drug therapy over the course of the 18 week experiment, and expect that a larger study with more mice per group might reveal statistically significant increases in CI-MPR. However, the statistically significant reduction in autophagosomes demonstrated by lower LC3-II indicated that the abnormal accumulation observed in Pompe disease were reduced by the addition of adjunctive β2 agonists during ERT. The lack of effect from gene therapy in the skeletal muscles can be attributed to lower efficiency of transduction with an AAV2/9 vector in skeletal muscle, in comparison with the heart.

[0107] The reduction of muscle glycogen content in absence of significantly increased GAA activity treatment has been reported, which is consistent with improved trafficking of GAA to the lysosomes following β2 agonist treatment. Furthermore, if the β2 agonist improved trafficking of CI-MPR-associated GAA to the lysosomes that could explain its beneficial effect upon glycogen content in absence of increased CI-MPR expression.

[0108] Overall, salmeterol has highly effective in comparison with the other drugs evaluated herein. Thus, salmeterol should be further developed as adjunctive therapy in combination with either ERT or gene therapy for Pompe disease.

[0109] Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore the spirit and scope of the appended claims should not be limited to the description and the preferred versions contained within this specification.

Claims

Claims

1. A method of treating a lysosomal storage disease in an individual comprising administering to the individual a therapeutically effective amount of a lysosomal enzyme and a therapeutic agent selected from growth hormone, an autocrine glycoprotein, a β2 agonist, or a combination thereof.

2. The method of claim 1, wherein the lysosomal storage disease is selected from Pompe disease, Gaucher disease, Fabry disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, or Niemann-Pick disease.

3. The method of claim 1, wherein the lysosomal enzyme is selected from glucocerebrosidase, acid alpha-glucosidase, alpha-galactosidase, alpha-n- acetylgalactosaminidase, acid sphingomyelinase, alpha-iduronidase, or a combination thereof.

4. The method of claim 1, wherein the therapeutic agent is a β2 agonist.

5. The method of claim 2, wherein the Pompe disease is selected from infantile Pompe disease, juvenile Pompe disease, or adult-onset Pompe disease.

6. The method of claim 3, wherein the acid alpha-glucosidase is selected from GAA, alglucosidase alfa, rhGAA, neo-rhGAA, reveglucosidase alpha, an rhGAA with higher M6P content than naturally found GAA that is administered with a chaperone, or a combination thereof.

7. The method of claim 4, wherein the β2 agonist is selected from albuterol, arbutamine, bambuterol, befunolol, bitolterol, bromoacetylalprenololmenthane, broxaterol, carbuterol, cimaterol, cirazoline, clenbuterol, clorprenaline, denopamine, dioxethedrine, dopexamine, ephedrine, epinephrine, etafedrine, ethylnorepinephrine, etilefrine, fenoterol, formoterol, hexoprenaline, higenamine, ibopamine, isoetharine, isoproterenol, isoxsuprine, mabuterol, metaproterenol, methoxyphenamine, norepinephrine, nylidrin, oxyfedrine, pirbuterol, prenalterol, procaterol, propranolol, protokylol, quinterenol, ractopamine, reproterol, rimiterol, ritodrine, salmefamol, soterenol, salmeterol, terbutaline, tretoquinol, tulobuterol, xamoterol, zilpaterol, zinterol, or a combination thereof.

8. The method of claim 1, wherein the therapeutic agent is administered bimonthly, monthly, biweekly, weekly, twice weekly, daily, twice a day, three times a day, or more often a day.

9. The method of claim 1, wherein the therapeutic agent is administered at a dose of about 20 μg per day to about 2100 μg per day.

10. The method of claim 1, wherein the lysosomal enzyme is administered bimonthly, monthly, biweekly, weekly, twice weekly, daily, twice a day, three times a day, or more often a day.

11. The method of claim 1, wherein the lysosomal enzyme is administered at a dose of about 1 mg/kg to about 50 mg per kg bodyweight of the individual.

12. The method of claim 1, wherein the therapeutic agent and the lysosomal enzyme are components of separate pharmaceutical compositions that are administered separately.

13. The method of claim 1, wherein the therapeutic agent and the lysosomal enzyme are components of separate pharmaceutical compositions that are mixed together before administration.

14. The method of claim 1, wherein the therapeutic agent is administered separately prior to, concurrently with, or subsequent to administration of the lysosomal enzyme.

15. The method of claim 1, wherein the therapeutic agent and the lysosomal enzyme are in a single pharmaceutical composition.

16. A method of increasing expression of a receptor for a lysosomal enzyme comprising administering a therapeutic agent as an adjunctive therapy to an individual who is undergoing or has undergone lysosomal enzyme replacement therapy.

17. A method of treating a lysosomal storage disorder in an individual in need thereof comprising administering to the individual a therapeutic agent as an adjunctive therapy to lysosomal enzyme replacement therapy.

18. The method of claim 17, wherein the lysosomal storage disorder is selected from Pompe disease, Gaucher disease, Fabry disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, or Niemann-Pick disease.

19. The method of claim 17, wherein the therapeutic agent is selected from a growth hormone, an autocrine glycoprotein, a β2 agonist, or a combination thereof.

20. The method of claim 17, wherein the lysosomal enzyme is selected from glucocerebrosidase, acid alpha-glucosidase, alpha-galactosidase, alpha-n- acetylgalactosaminidase, acid sphingomyelinase, alpha-iduronidase, or a combination thereof.