EA043909B1 - METHODS AND COMPOSITIONS FOR DELIVERY OF ARYLSULPHATASE A TO THE CNS - Google Patents

Description

Cross reference to related applications

This application claims priority under U.S. Provisional Patent Application No.

62/296563, filed February 17, 2016, and US Provisional Patent Application No. 62/453864, filed February 2, 2017; which are incorporated herein by reference in their entirety.

List of sequences

This application contains a sequence listing that was filed electronically and is incorporated herein by reference in its entirety. The file, named SHR1096_ST25.txt, was created on February 13, 2017 and is 8922 bytes in size.

BACKGROUND OF THE INVENTION

Enzyme replacement therapy (ERT) involves the systemic administration of natural or recombinantly produced proteins and/or enzymes to a subject. Approved therapies are typically administered intravenously to subjects and are generally effective in treating the physical symptoms of the underlying enzyme deficiency. Due to the limited distribution of intravenously administered protein and/or enzyme in cells and tissues of the central nervous system (CNS), the treatment of diseases characterized by a CNS-related etiology has been particularly problematic because intravenously administered proteins and/or enzymes do not cross the blood-brain barrier in sufficient concentrations ( BBB).

The blood-brain barrier (BBB) is a structural system composed of endothelial cells whose function is to protect the central nervous system (CNS) from harmful substances in the bloodstream, such as bacteria, macromolecules (such as proteins), and other hydrophilic molecules, by limiting the diffusion of such substances through the BBB and into the underlying cerebrospinal fluid (CSF) and CNS.

There are several ways to bypass the BBB to enhance delivery of a therapeutic to the brain, including direct intracranial injection, transient permeabilization of the BBB, and modification of the active substance to alter tissue distribution. Direct injection of a therapeutic agent into brain tissue allows complete bypass of the vasculature, but some difficulties arise with its use, mainly due to the risk of complications (infection, tissue damage, immunological reactivity) posed by intracranial injections and insufficient diffusion of the active substance from the injection site. To date, direct injection of proteins into the brain has not achieved significant therapeutic effect due to the presence of diffusion barriers and the limited volume of therapeutic agent that can be administered. Convective diffusion has been studied using catheters placed in the brain parenchyma using slow continuous infusions (Bobo et al., Proc. Natl. Acad. Sci. U.S.A 91, 2076-2080 (1994); Nguyen et al. J. Neurosurg. 98, 584-590 (2003)), but no approved therapies currently use this approach as long-term therapy. In addition, placement of intracerebral catheters is quite invasive and less desirable as a clinical alternative.

Intrathecal (IT) injection or cerebrospinal fluid (CSF) injection of proteins has also been attempted, but has not yet achieved therapeutic success. The main problem with this type of treatment is the tendency of the active substance to bind very tightly to the ependymal lining of the ventricle, which impedes subsequent diffusion. There are currently no approved products to treat genetic brain diseases by direct administration to the CSF.

In fact, many believed that the barrier to diffusion at the surface of the brain, as well as the lack of effective and convenient delivery methods, were too great a barrier to achieving adequate therapeutic effect in the brain for any disease.

Many lysosomal storage disorders affect the nervous system and therefore present special challenges in treating these diseases with traditional therapies. There is often significant accumulation of glycosaminoglycans (GAGs) in the neurons and meninges of affected individuals, leading to various forms of CNS symptoms. To date, none of the CNS symptoms caused by lysosomal disorders have been successfully treated by any available means.

Thus, there remains a significant need for efficient delivery of therapeutics to the brain. More specifically, there is a significant need for more efficient delivery of active substances to the central nervous system for the treatment of lysosomal storage disorders.

Brief description of the invention

The invention provides, among other things, compositions and methods for treating metachromatic leukodystrophy syndrome (MLD) by intrathecal delivery of arylsulfatase A.

In certain embodiments, methods are provided for treating metachromatic leukodystrophy (MLD) syndrome, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and

- 1 043909 with a certain interval between administrations for a period of treatment sufficient to improve, stabilize or reduce the rate of deterioration of one or more motor functions compared to the baseline.

In some embodiments, administration of the recombinant ASA enzyme further results in improvement, stabilization, or reduction in the rate of decline of one or more cognitive, adaptive, and/or executive functions.

In some embodiments, the one or more motor functions include a gross motor function. In some embodiments, gross motor function is assessed using a Gross Motor Functioning Test (GMFM), such as the GMFM-88. In some embodiments, the subject's raw GMFM-88 score is greater than 40%. In some embodiments, the patient's baseline GMFM-88 score is less than 40%. In some embodiments, administration of the recombinant ASA enzyme results in a reduction in the GMFM-88 score of less than 10, 20, 30, 40, or 50%. In some embodiments, administration of the recombinant ASA enzyme results in a significant stabilization of the GMFM-88 score. In some embodiments, administration of the recombinant ASA enzyme results in an improvement in the GMFM-88 score.

In certain embodiments, methods for treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and at some interval between administrations for a period of treatment sufficient to reduce levels biomarker that accumulates during MLD in physiological fluid selected from the group consisting of cerebrospinal fluid, urine, blood and serum, compared with baseline biomarker levels. In some embodiments, the biomarker is selected from the group consisting of sulfatide, lysosulfatide, and combinations thereof. In some embodiments, the biomarker is a sulfatide. In some embodiments, the physiological fluid is cerebrospinal fluid.

In some embodiments, the baseline level of sulfatides in the cerebrospinal fluid is greater than about 0.1 μg/mL. In some embodiments, the baseline level of sulfatides in the cerebrospinal fluid is greater than about 0.2 μg/mL. In some embodiments, the baseline cerebrospinal fluid sulfatide level is greater than about 0.3 μg/mL.

In some embodiments, administration of the recombinant ASA enzyme results in a decrease in cerebrospinal fluid sulfatide levels by greater than about 0.1 μg/mL. In some embodiments, administration of the recombinant ASA enzyme results in a decrease in cerebrospinal fluid sulfatide levels by greater than about 0.2 μg/mL.

In certain embodiments, methods of treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and at some interval between administrations for a period of treatment sufficient to increase levels biomarkers reduced in MLD in brain tissue compared with baseline biomarker levels.

In some embodiments, the brain tissue is deep white matter of the brain. In some embodiments, the biomarker is a metabolite, such as, for example, N-acetylaspartate. In some embodiments, N-acetyl aspartate levels are assessed using proton magnetic resonance spectroscopy.

In certain embodiments, methods for treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A enzyme at a therapeutically effective dose and at an interval between administrations for a period of treatment sufficient to stabilize or reduce the degree of involvement. in brain damage compared to baseline.

In some embodiments, brain damage involvement is assessed by an MLD severity score determined using MRI (magnetic resonance imaging). In some embodiments, administration of the recombinant ASA enzyme results in a decrease in the subject's MLD severity score, as determined by MRI, compared to baseline. In some embodiments, administration of the recombinant ASA enzyme results in stabilization of the subject's MLD severity score, as determined by MRI, compared to baseline.

In some embodiments, the therapeutically effective dose is or greater than 10 mg. In some embodiments, the therapeutically effective dose is or greater than 30 mg. In some embodiments, the therapeutically effective dose is or greater than 100 mg. In some embodiments, the therapeutically effective dose is less than 200 mg.

In some embodiments, the interval between administrations is one week. In some embodiments, the interval between administrations is two weeks. In some embodiments, the interval between administrations is one month.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In some embodiments, the subject's age is sixteen years or younger. In some embodiments, the subject's age is twelve years or less. In some embodiments, the subject's age is nine years or less. In some embodiments, the subject's age is six years or less. In some embodiments, the subject's age is four years or less. In some embodiments, the subject's age is three years or less. In some embodiments, the subject's age is two years or less. In some embodiments, the subject's age is 18 months or less. In some embodiments, the subject's age is 12 months or less.

In some embodiments, the subject's age is 6 months or less.

In some embodiments, the subject exhibits at least one symptom of metachromatic leukodystrophy. In some embodiments, the subject does not exhibit any symptoms of metachromatic leukodystrophy. In some embodiments, the subject is diagnosed with metachromatic leukodystrophy. In some embodiments, the subject is identified as being at risk for developing metachromatic leukodystrophy.

In some embodiments, arylsulfatase A is administered into the spinal canal. In some embodiments, arylsulfatase A is administered to the lumbar region. In some embodiments, arylsulfatase A is administered by lumbar puncture.

In some embodiments, intrathecal administration is accomplished through intermittent or continuous access to an implanted intrathecal drug delivery device (IDDD).

In some embodiments, the treatment period is at least 6 months. In some embodiments, the treatment period is at least 9 months. In some embodiments, the treatment period is at least 12 months. In some embodiments, the treatment period is at least 24 months. In some embodiments, the treatment period is at least 26 months.

In some embodiments, the subject does not experience serious adverse effects associated with administration of the recombinant arylsulfatase A.

In certain embodiments, recombinant arylsulfatase A (ASA) enzymes are provided for use in a method comprising the step of intrathecally administering to a subject at risk for or suffering from metachromatic leukodystrophy the recombinant arylsulfatase A enzyme at a therapeutically effective dose and at some interval between administrations over a period treatment sufficient to improve, stabilize, or reduce the rate of decline in one or more motor functions compared to baseline.

In certain embodiments, recombinant arylsulfatase A (ASA) enzymes are provided for use in a method comprising the step of intrathecally administering to a subject at risk for or suffering from metachromatic leukodystrophy the recombinant arylsulfatase A enzyme at a therapeutically effective dose and at some interval between administrations over a period treatment sufficient to reduce cerebrospinal fluid (CSF) sulfatide levels from baseline.

In certain embodiments, recombinant arylsulfatase A (ASA) enzymes are provided for use in a method comprising the step of intrathecally administering to a subject at risk for or suffering from metachromatic leukodystrophy the recombinant arylsulfatase A enzyme at a therapeutically effective dose and at some interval between administrations over a period treatment sufficient to stabilize or reduce the extent of brain injury involvement compared to baseline.

In certain embodiments, routes are provided for the use of recombinant arylsulfatase A enzymes in the manufacture of a medicament for the treatment or prevention of metachromatic leukodystrophy, wherein the treatment comprises the step of intrathecally administering to a subject at risk for or suffering from metachromatic leukodystrophy the recombinant arylsulfatase A enzyme at a therapeutically effective dose and with at some interval between administrations for a period of treatment sufficient to improve, stabilize, or reduce the rate of deterioration of one or more motor functions compared to baseline.

In certain embodiments, routes are provided for the use of recombinant arylsulfatase A enzymes in the manufacture of a medicament for the treatment or prevention of metachromatic leukodystrophy, wherein the treatment comprises the step of intrathecally administering to a subject at risk for or suffering from metachromatic leukodystrophy the recombinant arylsulfatase A enzyme at a therapeutically effective dose and with some interval between doses for a treatment period sufficient to reduce cerebrospinal fluid (CSF) sulfatide levels from baseline.

In certain embodiments, routes are provided for the use of recombinant arylsulfatase A enzymes in the manufacture of a medicament for the treatment or prevention of metachromatic leukodystrophy, wherein the treatment comprises the step of intrathecally administering to a subject at risk for or suffering from metachromatic leukodystrophy the recombinant arylsulfatase A enzyme at a therapeutically effective dose and with some interval between administrations for a period of treatment sufficient to stabilize or reduce the extent of brain damage involvement compared to baseline.

In some embodiments, the recombinant ASA enzymes contain an amino acid sequence that is at least 85% identical at the amino acid level to SEQ ID NO: 1. In some embodiments, the recombinant ASA enzymes contain an amino acid sequence that is at least 90% identical at the amino acid level to SEQ ID NO: 1. In some embodiments, the recombinant ASA enzymes contain an amino acid sequence that is at least 95% identical at the amino acid level to SEQ ID NO: 1.

In some embodiments, the recombinant ASA enzymes contain an amino acid sequence that is at least 98% identical at the amino acid level to SEQ ID NO: 1. In some embodiments, the recombinant ASA enzymes contain the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the recombinant ASA enzymes contain an amino acid sequence containing no more than four mismatches from SEQ ID NO: 1. In some embodiments, the recombinant ASA enzymes contain an amino acid sequence containing no more than three mismatches from SEQ ID NO: 1. In some embodiments recombinant ASA enzymes contain an amino acid sequence containing no more than two mismatches with SEQ ID NO: 1.

In some embodiments, the recombinant ASA enzymes contain an amino acid sequence containing no more than one mismatch to SEQ ID NO: 1.

Brief description of graphic materials

In fig. Figure 1 illustrates the design of a non-randomized, open-label, 40-week phase 1/2 dose-escalation clinical trial of intrathecally delivered recombinant human arylsulfatase A in children with metachromatic leukodystrophy (MLD). IDDD: intrathecal drug delivery device; rhASA: recombinant human arylsulfatase A.

In fig. Figure 2 shows the change in Gross Motor Functioning Test-88 (GMFM-88) total score (from baseline) as a percentage in each of the three treatment groups (10, 30, and 100 mg rhASA) at week 40 of the phase 1/2 clinical trial. described in Example 1. Data shown are least squares means; Error bars represent standard error. Raw data are presented as mean ± standard deviation. Comparisons between groups were analyzed using analysis of covariance (ANCOVA) with changes in GMFM-88 score from baseline as the dependent variable and dose group and baseline as covariates.

In fig. Figure 3 shows the treatment effect (measured by change from baseline in GMFM-88 total score (%)) compared to baseline GMFM-88 total score (%) in patients in the clinical trial described in Example 1. Circles: 10 mg rhASA; squares: 30 mg rhASA; diamonds: 100 mg rhASA.

In fig. Figure 4 shows the change in GMFM-88 total score (%) across all treatment groups in the clinical trial described in Example 1, with values for those patients who had baseline GMFM-88 total scores greater than 40% presented in separate columns. Solid black bars show data for all patients, and solid white bars show data only for patients with baseline GMFM-88 total scores greater than 40%. Data in the figures are presented as means, with error bars indicating standard deviation values. Raw data are presented as mean ± standard deviation.

In fig. 5 shows the change in the MRI severity score for metachromatic leukodystrophy at week 40 in patients enrolled in the clinical study described in Example 1. Data shown are least squares means; Error bars represent standard error. Raw data are presented as mean ± standard deviation. Correction for unbalanced raw values was made by using the least squares mean.

In fig. 6 shows cerebrospinal fluid sulfatide levels at week 40 in patients enrolled in the clinical trial described in Example 1. The data shown are presented in

- 4 043909 as average values; Error bars represent standard deviation. Raw data are presented as mean ± standard deviation.

In fig. Figure 7 shows CSF sulfatide levels (in μg/mL) over time by cohort in patients enrolled in the clinical trial. The data includes data from at least two sibling pairs. Data are shown for patients administered 10 mg, 30 mg, and 100 mg of recombinant human arylsulfatase A.

In fig. Figure 8 illustrates the design of an open-label, single-arm, long-term, extension phase 1/2 clinical trial of intrathecally delivered recombinant human arylsulfatase A in children with metachromatic leukodystrophy (MLD).

In fig. 9A and 9B demonstrate the effect of intrathecally delivered rhASA on motor function over 104 weeks of treatment. In fig. Figure 9A shows the mean GMFM-88 total scores for each cohort across study visits. In fig. Figure 9B shows individual GMFM-88 total scores by patient age over 104 weeks of treatment.

In fig. 10 shows the effect of intrathecally delivered rhASA on MRI severity score of MLD after 104 weeks of treatment. The baseline score is shown as the mean ± standard deviation of the total MRI severity score of MLD at baseline (score ranges 0 to 34, with higher scores indicating greater disease severity).

In fig. 11A and 11B show the effect of intrathecally delivered rhASA on the mean NAA/creatine metabolite ratio in brain white matter over 104 weeks of treatment. In fig. 11A shows the white matter of the right frontal lobe, and FIG. Figure 11B shows the white matter of the right frontal and parietal lobes. Data are shown as mean ± standard error.

In fig. 12 shows the effect of intrathecally delivered rhASA on CSF sulfatide concentrations over 104 weeks of treatment. Data are shown as mean ± standard deviation.

Definitions

To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this specification.

Approximately or approximately. As used herein, the term approximately or approximately, when applied to one or more values of interest, refers to a value similar to a specified reference value. In certain embodiments, the term about or approximately refers to a range of values within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less in any direction (more or less) from the specified reference value, unless otherwise stated or otherwise obvious from the context (unless such number would exceed 100% of the possible value).

Reduce intensity. As used herein, the term amelioration refers to preventing, reducing the intensity of symptoms, or weakening or improving the condition of a subject. Reduction involves, but does not require, complete recovery from a disease state or its complete prevention. In some embodiments, the decrease in intensity includes an increase in the levels of the corresponding protein or its activity that is deficient in the corresponding affected tissues.

Initial level. As used herein, the term baseline refers to a value, level, status, condition, etc. before the start of a treatment period, typically at the start of treatment.

Biologically active. As used herein, the phrase biologically active refers to the characteristic of any agent having activity in a biological system, and in particular in the body. For example, a drug that, when administered to an organism, produces a biological effect on that organism is considered biologically active. In specific embodiments, in which the protein or polypeptide is biologically active, the portion of the protein or polypeptide that has at least one biological activity of the protein or polypeptide is generally referred to as the biologically active portion.

Volume-forming agent. As used herein, the term bulking agent refers to a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (eg, facilitates the production of a substantially uniform lyophilized cake that maintains an open-cell structure). Exemplary bulking agents include mannitol, glycine, sodium chloride, hydroxyethyl starch, lactose, sucrose, trehalose, polyethylene glycol and dextran.

Cation-independent mannose-6-phosphate receptor (CI-MPR). As used herein, the term cation-independent mannose-6-phosphate receptor (CI-MPR) refers to a cellular receptor that binds mannose-6-phosphate (M6P) marks on acid hydrolase precursors in the apparatus

- 5 043909

Golgi, intended for transport to lysosomes. In addition to mannose-6-phosphates, CI-MPR also binds other proteins, including IGF-II. CI-MPR is also known as M6P/IGF-II receptor, CI-MPR/IGF-II receptor, IGF-II receptor, or IGF2 receptor. These terms and their abbreviations are used interchangeably in this document.

Concomitant immunosuppressive therapy. As used herein, the term concomitant immunosuppressive therapy includes any immunosuppressive therapy administered as pretreatment, preconditioning, or concurrently with a treatment regimen.

Diluent. As used herein, the term diluent refers to a pharmaceutically acceptable (eg, safe and non-toxic when administered to humans) diluent useful in preparing a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

Dosage form. As used herein, the terms dosage form and unit dosage form refer to a physically discrete unit of therapeutic protein for a patient to be treated. Each unit contains a predetermined amount of active material calculated to obtain the desired therapeutic effect. However, it will be understood that the decision regarding the total dosage of the composition will be made by the attending physician based on a thorough medical evaluation.

Enzyme replacement therapy (ERT). As used herein, the term enzyme replacement therapy (ERT) refers to any therapeutic strategy that corrects an enzyme deficiency by providing the missing enzyme. In some embodiments, the missing enzyme is provided by intrathecal administration. In some embodiments, the missing enzyme is provided by infusion into the bloodstream. Once administered, the enzyme is taken up by the cells and transported to the lysosomes, where the enzyme acts to eliminate material accumulated in the lysosomes due to enzyme deficiency. Typically, for lysosomal enzyme replacement therapy to be effective, the therapeutic enzyme is delivered to the lysosomes in the appropriate cells in the target tissues where the storage defect occurs.

Improve, increase or decrease. As used herein, the terms improve, increase, or decrease, or their grammatical equivalents, indicate values 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 controls). individuals) in the absence of treatment described herein. A control individual is an individual affected by the same form of lysosomal storage disease as the treated individual, who is approximately the same age as the treated individual (to ensure that the stages of the disease in the treated and control individual( s) of the individual(s) are comparable).

Individual, subject, patient. As used herein, the terms subject, individual, or patient refer to a human or non-human mammalian subject. An individual (also called a patient or subject) being treated is an individual (fetus, infant, child, adolescent or adult) suffering from a disease.

Intrathecal administration. As used herein, the term intrathecal administration or intrathecal injection refers to an injection into the spinal canal (the intrathecal space surrounding the spinal cord). Various techniques can be used, including lumbar puncture. In some embodiments, intrathecal administration or intrathecal delivery in accordance with the present invention refers to IT administration or delivery to the lumbar region or region, i.e. to lumbar IT insertion or delivery. As used herein, the term lumbar region or lumbar region refers to the area between the third and fourth lumbar vertebrae (in the lower back) and, more meaningfully, the L2-S1 region of the spine.

Linker. As used herein, the term linker refers to an amino acid sequence in a fusion protein that is different from that present at a particular position in the native protein and is typically designed to be flexible, or to be incorporated into a structure, such as a helix, between two protein components. The linker is also called a spacer.

Lyoprotector. As used herein, the term lyoprotectant refers to a molecule that prevents or reduces the chemical and/or physical instability of a protein or other substance during lyophilization and subsequent storage. Exemplary lyoprotectants include sugars such as sucrose or trehalose; an amino acid such as monosodium glutamate or histidine; methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher sugar alcohols, for example glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; pluronics and their combinations. In some embodiments, the lyoprotectant is a non-reducing sugar such as trehalose or sucrose.

Lysosomal enzyme. As used herein, the term lysosomal enzyme refers to any enzyme that is capable of reducing the amount of storage material in mammalian lysosomes or can provide relief or reduction in the severity of one or more symptoms of a lysosomal storage disease. Lysosomal enzymes suitable for the present invention include both wild-type and modified lysosomal enzymes and can be produced by recombinant and synthetic methods or purified from natural sources. Examples of lysosomal enzymes are listed in Table. 1.

Deficiency of lysosomal enzymes. As used herein, lysosomal enzyme deficiency refers to a group of genetic disorders caused by a deficiency of at least one of the enzymes required to break down macromolecules (eg, enzyme substrates) into peptides, amino acids, monosaccharides, nucleic acids and fatty acids in lysosomes. As a result, individuals suffering from various forms of lysosomal enzyme deficiency accumulate materials in various tissues (eg, the central nervous system, liver, spleen, intestines, blood vessel walls, and other organs).

Lysosomal storage disease. As used herein, the term lysosomal storage disease refers to any disease caused by a deficiency of one or more lysosomal enzymes necessary for the transformation of natural macromolecules during metabolism. These diseases typically result in the accumulation of undegraded molecules in lysosomes, resulting in an increase in the number of storage granules (also called storage vesicles). These diseases and various examples are described in more detail below.

Polypeptide. As used herein, a polypeptide, generally speaking, is a string of at least two amino acids linked together by a peptide bond. In some embodiments, the polypeptide may contain at least 3-5 amino acids, each of which is linked to the others by at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides do not necessarily sometimes contain unnatural amino acids or other entities that are nonetheless capable of being incorporated into the polypeptide chain.

Replacement enzyme. As used herein, the term replacement enzyme refers to any enzyme that can act to at least partially replace a deficient or missing enzyme in a disease being treated. In some embodiments, the term replacement enzyme refers to any enzyme that can act to at least partially replace a deficient or missing lysosomal enzyme in a lysosomal storage disease being treated. In some embodiments, the replacement enzyme is capable of reducing the amount of material that accumulates in mammalian lysosomes or can provide relief or reduction in the severity of one or more symptoms of a lysosomal storage disease. Replacement enzymes suitable for the present invention include both wild-type and modified lysosomal enzymes and can be produced by recombinant and synthetic methods or purified from natural sources. The replacement enzyme may be a recombinant, synthetic, gene-activated or naturally occurring enzyme.

Soluble. As used herein, the term soluble refers to the ability of a therapeutic agent to form a homogeneous solution. In some embodiments, the solubility of the therapeutic agent in the solution into which it is administered and carried to the target site of action (eg, brain cells and tissues) is sufficient to ensure delivery of a therapeutically effective amount of the therapeutic agent to the target site of action. The solubility of therapeutic agents can be influenced by several factors. For example, significant factors that can influence protein solubility include ionic strength, amino acid sequence, and the presence of other co-solubilizing agents or salts (eg, calcium salts). In some embodiments, the pharmaceutical compositions are formulated such that calcium salts are not included in such compositions. In some embodiments, the therapeutic agents of the present invention are soluble in their respective pharmaceutical composition. It will be understood that although isotonic solutions are generally preferred for parenteral administration of drugs, the use of isotonic solutions may limit the proper solubility for some therapeutic agents, and in particular for some proteins and/or enzymes. Mildly hypertonic solutions (eg, up to 175 mM sodium chloride in 5 mM sodium phosphate at pH 7.0) and sugar-containing solutions (eg, up to 2% sucrose in 5 mM sodium phosphate at pH 7.0) have been demonstrated to be well tolerated by monkeys . For example, the most common approved CNS bolus formulation is saline (150 mM NaCl in water).

- 7 043909

Stability. As used herein, the term stable refers to the ability of a therapeutic agent (eg, a recombinant enzyme) to maintain its therapeutic effectiveness (eg, all or substantially all of its intended biological activity and/or physicochemical integrity) over extended periods of time. The stability of a therapeutic agent and the ability of a pharmaceutical composition to maintain the stability of such a therapeutic agent can be assessed over extended periods of time (eg, for at least 1, 3, 6, 12, 18, 24, 30, 36 months or longer). In general, the pharmaceutical compositions described herein have been formulated so that they are capable of stabilizing one or more therapeutic agents with which they are formulated (eg, recombinant proteins), or, alternatively, slowing down or preventing their degradation. With respect to formulation, a stable formulation is one in which the therapeutic agent contained therein effectively retains its physical and/or chemical integrity and biological activity during storage and processes (such as freezing/thawing, mechanical mixing and lyophilization). An indicator of protein stability can be the formation of high molecular weight (HMW) aggregates, loss of enzymatic activity, formation of peptide fragments, and shifts in charge distribution profiles.

Subject. As used herein, the term subject means any mammal, including humans. In certain embodiments of the present invention, the subject is an adult, adolescent, child, or infant. The present invention also covers the administration of pharmaceutical compositions and/or methods of treatment in utero.

Substantially homologous. The phrase substantially homologous is used herein to refer to comparisons between amino acid sequences or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be substantially homologous if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues having correspondingly similar structural and/or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as hydrophobic or hydrophilic amino acids and/or as having polar or nonpolar side chains. Replacing one amino acid with another of the same type can often be considered a homologous substitution.

As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a number of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gap BLAST and PSI-BLAST for amino acid sequences . Such exemplary programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Bid., 215(3): 403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree homology. In some embodiments, two sequences are considered substantially homologous if at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99% or more of their corresponding residues are homologous over a significant portion of the residues. In some embodiments, the significant fragment is the entire sequence. In some embodiments, the significant fragment has at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more balances.

Pretty much identical. The phrase substantially identical is used herein to refer to comparisons between amino acid sequences or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered substantially identical if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a number of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gap BLAST and PSI-BLAST for amino acid sequences . Such exemplary programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Bid., 215(3): 403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identification

- 8 043909 tion of identical sequences, the programs mentioned above generally provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99% or more of their corresponding residues are identical over a significant portion of the residues. In some embodiments, the significant fragment is the entire sequence. In some embodiments, the significant fragment has at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more balances.

Synthetic CSF. As used herein, the term synthetic CSF refers to a solution having the pH, electrolyte composition, glucose content and osmolarity corresponding to cerebrospinal fluid. Synthetic CSF is also called artificial CSF. In some embodiments, the synthetic CSF is Elliott B solution.

Suitable for delivery to the central nervous system. As used herein, the phrase suitable for CNS delivery or suitable for intrathecal delivery associated with the pharmaceutical compositions of the present invention generally refers to the stability, tolerability and solubility properties of such compositions, as well as the ability of such compositions to deliver an effective amount of the therapeutic agent contained in them to the target site of delivery (eg, CSF or brain).

Target tissues. As used herein, the term target tissue refers to any tissue affected by a lysosomal storage disease to be treated, or any tissue that normally expresses a deficient lysosomal enzyme. In some embodiments, target tissues include those tissues that have detectable or abnormally high amounts of enzyme substrate, such as stored in cellular lysosomes of the tissue, in patients suffering from or susceptible to lysosomal storage disease. In some embodiments, target tissues include those tissues that exhibit a disease process, symptom, or sign associated with a disease. In some embodiments, target tissues include those tissues in which the deficient lysosomal enzyme is normally expressed at elevated levels. As used herein, the target tissue may be a brain target tissue, a spinal cord target tissue, and/or a peripheral target tissue. Exemplary target tissues are described in detail below.

Therapeutic component. As used herein, the term therapeutic moiety refers to the portion of a molecule that imparts a therapeutic effect to the molecule. In some embodiments, the therapeutic component is a polypeptide having therapeutic activity.

A therapeutically effective amount. As used herein, the term therapeutically effective amount refers to an amount of therapeutic protein (eg, enzyme replacement) that provides a therapeutic effect to the subject being treated at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measured by a specific test or marker) or subjective (i.e., the subject indicates or perceives the effect). In particular, a therapeutically effective amount refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventive effect, such as by reducing the severity of symptoms associated with a disease, preventing or delaying the onset of disease manifestation and/or also reducing the severity or frequency of disease symptoms. A therapeutically effective amount is typically administered according to a dosage regimen, which may include multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and/or the appropriate unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration, combination with other pharmaceutical agents. In addition, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical used; the specific composition used; age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the particular fusion protein used; duration of treatment and similar factors well known in the medical field.

Portable. As used herein, the terms tolerable and tolerable refer to the ability of the pharmaceutical compositions of the present invention not to cause an adverse reaction in a subject to whom such a composition is administered, or, alternatively, not to cause a serious adverse reaction in a subject to which such a composition is administered. In some embodiments, the pharmaceutical compositions of the present invention are well tolerated by the subject to whom such compositions are administered.

- 9 043909

Treatment. As used herein, the term treatment (also treat or treat) refers to any administration of a therapeutic protein (e.g., a lysosomal enzyme) that results in partial or complete relief, amelioration, attenuation, inhibition, delayed onset, reduction in severity, and/or reducing the incidence of one or more symptoms or signs of a specific disease, disorder and/or condition (eg, metachromatic leukodystrophy). Such treatment may be administered to a subject who is not showing signs of the corresponding disease, disorder and/or condition, and/or to a subject who is showing only early signs of the disease, disorder and/or condition. Alternatively or additionally, such treatment may be administered to a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.

Detailed Description of the Invention

The present invention provides, among other things, improved methods and compositions for effective direct delivery of a therapeutic agent to the central nervous system (CNS). The present invention is based on the unexpected discovery that a replacement enzyme (eg, ASA protein) for a lysosomal storage disease (eg, metachromatic leukodystrophy disease) can be administered directly into the cerebrospinal fluid (CSF) of a subject in need of treatment at a high concentration without inducing significant adverse effects in the subject. More surprisingly, the present inventors have discovered that the replacement enzyme can be delivered in a simple saline or buffer based formulation without the use of synthetic CSF. Even more surprisingly, intrathecal delivery in accordance with the present invention does not result in significant undesirable effects, such as a strong immune response, in the subject. Thus, in some embodiments, intrathecal delivery in accordance with the present invention can be used in the absence of concomitant immunosuppressive therapy (eg, without induction of immune tolerance by pretreatment or preconditioning).

In some embodiments, intrathecal delivery in accordance with the present invention allows for effective diffusion into various brain tissues, resulting in effective delivery of the replacement enzyme to various target brain tissues in superficial, shallow and/or deep brain regions. In some embodiments, intrathecal delivery in accordance with the present invention results in the delivery of sufficient amounts of replacement enzymes to the peripheral portion of the circulatory system. As a result, in some cases, intrathecal delivery in accordance with the present invention results in delivery of the replacement enzyme to peripheral tissues such as the liver, heart, spleen and kidneys. This finding is unexpected and may be particularly applicable to the treatment of lysosomal storage diseases that have both a CNS and a peripheral component, which will typically require both regular intrathecal and intravenous administration. It is believed that intrathecal delivery in accordance with the present invention may allow a reduction in the dose and/or frequency of IV injection without negatively affecting the therapeutic effects in the treatment of peripheral symptoms.

The present invention provides various unexpected and useful features that allow efficient and convenient delivery of enzyme replacement to various target brain tissues, resulting in effective treatment of lysosomal storage diseases having CNS features.

Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may apply to any aspect of the present invention. In this application, the use of or means and/or, unless otherwise indicated.

Recombinant arylsulfatase A enzymes.

In some embodiments, the methods and compositions of the present invention are used to deliver recombinant arylsulfatase A (ASA) enzyme to the CNS for the treatment of the disease metachromatic leukodystrophy. A suitable ASA enzyme can be any molecule or part of a molecule that can replace the activity of the naturally occurring arylsulfatase A (ASA) enzyme or provide relief from one or more phenotypes or symptoms associated with ASA deficiency. In some embodiments, a replacement enzyme useful for the present invention is a polypeptide having an N-terminus and a C-terminus and an amino acid sequence substantially similar to or identical to that of the mature human ASA enzyme.

Typically, human ASA is produced as a precursor molecule that is processed to its mature form. This process typically occurs through the removal of an 18-amino acid signal peptide. Generally, the precursor shape is also called full-length precursor

- 10 043909 the parent or full-length ASA enzyme, which contains 507 amino acids. The 18 N-terminal amino acids are cleaved off, resulting in a mature form of 489 amino acids in length. Thus, it is believed that the N-terminal 18 amino acids are not generally required for ASA enzyme activity. The amino acid sequences of the mature form (SEQ ID NO: 1) and full-length precursor (SEQ ID NO: 2) of an exemplary wild-type or naturally occurring human ASA enzyme are shown in Table 1. 1.

Table 1

Human ASA Sequence

Mature form RPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFY

VPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVA EVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQ GPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLE ARYMAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSG RGPFGDSLMELDAAVGTLMTAIGDL GLLEETLVIFTADNGPETMR MSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASS LDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPS YPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAH EPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDA AVTFGPSQVARGE DPALQICCHPGCTPRPACCHCPDPHA (SEQ ID NO: 1)

Full size MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGDLGCYGHPSST predecessor TPNLDQLAAGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPG VLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFL PPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIP LLANLSVEAQPPWLPGLEAR YMAFAHDLMADAQRQDRPFFLYYAS HHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGL LEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPAL AFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSP LLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAH SDTTADPA CHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATP EVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTP RPACCHCPDPHA (SEQ ID NO: 2)

Thus, in some embodiments, the recombinant arylsulfatase A of the present invention is a mature human ASA protein (SEQ ID NO: 1). In some embodiments, a suitable recombinant arylsulfatase A may be a homologue or analogue of the mature human ASA protein. For example, a homolog or analogue of a mature human ASA protein may be a modified mature human ASA protein containing one or more amino acid substitutions, deletions and/or insertions relative to a wild-type or naturally occurring ASA protein (e.g., SEQ ID NO: 1) and at the same time retaining to a significant extent the activity of the ASA protein. Thus, in some embodiments, the recombinant arylsulfatase A useful for the present invention is substantially homologous to the mature human ASA protein (SEQ ID NO: 1). In some embodiments, a recombinant arylsulfatase A suitable for the present invention has an amino acid sequence of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99% or more homologous to SEQ ID NO: 1. In some embodiments, the recombinant arylsulfatase A useful for the present invention is substantially identical to the mature human ASA protein (SEQ ID NO: 1). In some embodiments, a recombinant arylsulfatase A suitable for the present invention has an amino acid sequence of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99% or more identical to SEQ ID NO: 1. For example, in some embodiments, the recombinant arylsulfatase A enzyme contains an amino acid sequence that is at least 85% identical at the amino acid level to SEQ ID NO: 1. In some embodiments, the recombinant ASA enzyme contains an amino acid sequence that is at least 90% identical at the amino acid level to SEQ ID NO: 1. In some embodiments, the recombinant arylsulfatase A enzyme contains an amino acid sequence that is at least 95% identical at the amino acid level to SEQ ID NO: 1 In some embodiments, the recombinant arylsulfatase A enzyme comprises an amino acid sequence that is at least 98% identical at the amino acid level to SEQ ID NO: 1. In some embodiments, the recombinant arylsulfatase A enzyme comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the recombinant arylsulfatase A enzyme contains an amino acid sequence having a small number of mismatches with SEQ ID NO: 1, such as no more than four, no more than three, no more than two, or no more than one mismatch with SEQ ID NO:

- 11 043909

1.

In some embodiments, the recombinant arylsulfatase A suitable for the present invention contains a fragment or portion of a mature human ASA protein. In some embodiments, the moiety or portion is catalytically active.

Additionally or alternatively, a replacement enzyme suitable for the present invention is a full-length ASA protein. In some embodiments, a suitable replacement enzyme may be a homolog or analogue of the full-length human ASA protein. For example, a homolog or analog of a full-length human ASA protein may be a modified full-length human ASA protein containing one or more amino acid substitutions, deletions and/or insertions relative to wild-type or naturally occurring full-length ASA protein (e.g., SEQ ID NO : 2) and while largely retaining the activity of the ASA protein. Thus, in some embodiments, a replacement enzyme useful for the present invention is substantially homologous to the full-length human ASA protein (SEQ ID NO: 2). In some embodiments, a replacement enzyme suitable for the present invention has an amino acid sequence of at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more homologous to SEQ ID NO: 2. In some embodiments, a replacement enzyme suitable for the present invention is substantially identical to SEQ ID NO: 2. In some embodiments, a replacement enzyme suitable for the present invention is has an amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO : 2. In some embodiments, a replacement enzyme suitable for the present invention comprises a fragment or portion of a full-length human ASA protein. As used herein, a full-length ASA protein typically contains a signal peptide sequence.

In some embodiments, the recombinant arylsulfatase A enzyme comprises a targeting moiety (eg, a lysosome targeting sequence) and/or a membrane permeating peptide. In some embodiments, the targeting sequence and/or membrane-permeable peptide is an integral part of the recombinant arylsulfatase A (eg, through a chemical linkage, through a fusion protein). In some embodiments, the targeting sequence comprises a mannose-6-phosphate moiety. In some embodiments, the targeting sequence comprises an IGF-I component. In some embodiments, the targeting sequence comprises an IGF-II component.

In some embodiments, a recombinant arylsulfatase A enzyme suitable for the present invention may have a wild-type or naturally occurring sequence. In some embodiments, a recombinant arylsulfatase A enzyme suitable for the present invention may have a modified sequence that is substantially homologous or identical to the wild type or naturally occurring sequence (e.g., having at least 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 98% sequence identity with wild type or naturally occurring sequence).

Replacement enzymes (eg, recombinant arylsulfatase A enzyme) suitable for the present invention can be produced recombinantly, for example, by using a host cell system engineered to express a nucleic acid encoding the recombinant enzyme. Alternatively or additionally, replacement enzymes can be produced in part or in whole by chemical synthesis.

In cases where the replacement enzyme is produced recombinantly, any expression system can be used. As just a few examples, known expression systems include, for example, eggs, baculovirus-infected cells, insect, plant, yeast or mammalian cells.

In some embodiments, enzymes suitable for the present invention are produced in mammalian cells. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include the murine myeloma cell line BALB/c (NSO/1, ECACC No. 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney cell line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); a human embryonic kidney cell line (293 or 293 cells subcultured for growth, Graham et al., J. Gen Virol., 36:59,1977); human fibrosarcoma cell line (eg, HT1080); newborn hamster kidney cells (BHK, ATCC CCL 10); Chinese Hamster Ovary +/-DHFR cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); liver cells

- 12 043909 people (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51);

TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells and a human hepatoma cell line (Hep G2).

In some embodiments, the methods of the present invention are used to deliver replacement enzymes produced in human cells. In some embodiments, the methods of the present invention are used to deliver replacement enzymes produced in CHO cells.

In some embodiments, replacement enzymes delivered by the method of the present invention contain a component that binds to a receptor on the surface of brain cells to facilitate cellular uptake and/or targeting to lysosomes. For example, such a receptor may be the cation-independent mannose-6-phosphate receptor (CIMPR), which binds mannose-6-phosphate (M6P) residues. In addition, CI-MPR also binds other proteins, including IGF-II. In some embodiments, a replacement enzyme suitable for the present invention contains M6P residues on the surface of the protein. In some embodiments, a replacement enzyme suitable for the present invention may contain bisphosphorylated oligosaccharides having higher binding affinity to CI-MPR. In some embodiments, a suitable enzyme contains up to an average of at least about 20% bisphosphorylated oligosaccharides per enzyme. In other embodiments, a suitable enzyme may contain approximately 10, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60% bisphosphorylated oligosaccharides per enzyme. Although such bisphosphorylated oligosaccharides may be naturally present in an enzyme, it should be noted that enzymes can be modified to contain such oligosaccharides. For example, suitable replacement enzymes can be modified with certain enzymes capable of catalyzing the transfer of N-acetylglucosamine-L-phosphate from UDP-GlcNAc to the 6' position of α-1,2-linked mannose residues in lysosomal enzymes. Methods and compositions for the preparation and use of such enzymes are described, for example, by Canfield et al. in US Pat. No. 6,537,785 and US Pat. No. 6,534,300, each of which is incorporated herein by reference.

In some embodiments, replacement enzymes for use in the present invention may be conjugated or fused to a lysosome-targeting moiety that is capable of binding to a receptor on the surface of brain cells. Suitable lysosome-targeting moieties may include IGF-I, IGF-II, RAP, p97, and variants, homologs, or fragments thereof (e.g., including a peptide having the sequence of at least 70, 75, 80, 85, 90 or 95% identical to the sequence of the mature human peptide IGF-I, IGF-II, RAP, wild-type p97).

In some embodiments, replacement enzymes useful in the present invention have not been modified to enhance delivery or transport of such agents through the BBB and into the CNS.

In some embodiments, the therapeutic protein comprises a targeting moiety (eg, a lysosome targeting sequence) and/or a membrane permeating peptide. In some embodiments, the targeting sequence and/or membrane-permeable peptide is an integral part of the recombinant arylsulfatase A (eg, through a chemical linkage, through a fusion protein). In some embodiments, the targeting sequence comprises a mannose-6-phosphate moiety. In some embodiments, the targeting sequence comprises an IGF-I component. In some embodiments, the targeting sequence comprises an IGF-II component.

Compositions.

Aqueous pharmaceutical solutions and compositions (i.e., formulations) traditionally used to deliver therapeutic agents to the CNS of a subject include unbuffered isotonic saline and Elliott's B solution, which is an artificial CSF. A comparison showing the composition of CSF compared to the composition of Elliott B solution is contained in Table. 2 below. As shown in table. 2, the concentration of Elliott's solution B closely matches the concentration of CSF. Elliott's solution B, however, has a very low buffer concentration and, accordingly, may not provide the adequate buffering capacity necessary to stabilize therapeutics (eg, proteins), particularly over extended periods of time (eg, under storage conditions). In addition, Elliott B solution contains certain salts that may be incompatible with formulations intended to deliver certain therapeutic agents, and in particular proteins or enzymes. For example, calcium salts present in Elliott B solution can mediate protein precipitation and thus reduce the stability of the formulation.

- 13 043909

table 2

Solution Na ⁺ meq/l K ⁺ meq/ Ca ⁺⁺ meq/ MD ⁺⁺ meq/ NHS- mEq/ Clmeq/ pH Phospho P mg/l Gluko for mg/l CSF 117- 137 2.3 2.2 2.2 22, 9 IZ127 7.31 1,2- 2.1 45-80 Elliott solution B 149 2.6 2.7 2.4 22, 6 132 6.0- 7.5 2.3 80

The present invention provides compositions in aqueous form, pre-lyophilization form, lyophilized form or diluted form for therapeutic agents that are formulated to be capable of stabilizing or, alternatively, retarding or preventing the degradation of one or more therapeutic agents with which they are composed (eg recombinant proteins). In some embodiments, the compositions of the present invention provide a lyophilized formulation for therapeutic agents. In some embodiments, the compositions of the present invention provide aqueous formulations for therapeutic agents. In some embodiments, the formulations are stable formulations.

Stable compositions.

As used herein, the term stable refers to the ability of a therapeutic agent (eg, a recombinant enzyme) to maintain its therapeutic efficacy (eg, all or substantially all of its intended biological activity and/or physicochemical integrity) over extended periods of time. The stability of a therapeutic agent and the ability of a pharmaceutical composition to maintain the stability of such a therapeutic agent can be assessed over extended periods of time (eg, preferably for at least 1, 3, 6, 12, 18, 24, 30, 36 months or longer). With respect to formulation, a stable formulation is one in which the therapeutic agent contained therein effectively retains its physical and/or chemical integrity and biological activity during storage and processes (such as freezing/thawing, mechanical mixing and lyophilization). An indicator of protein stability can be the formation of high molecular weight (HMW) aggregates, loss of enzymatic activity, formation of peptide fragments, and shifts in charge distribution profiles.

The stability of a therapeutic agent is particularly important with respect to maintaining the specified concentration range of the therapeutic agent required to ensure that the agent performs its intended therapeutic function. The stability of a therapeutic agent can be further assessed in terms of the biological activity or physicochemical integrity of the therapeutic agent over extended periods of time. For example, stability at a given time point can be compared with stability at an earlier time point (eg, day 0 after formulation) or with an unformulated therapeutic agent, and the results of this comparison expressed as a percentage. The pharmaceutical compositions of the present invention preferably retain at least 100%, at least 99%, at least 98%, at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% of the biological activity or physicochemical integrity of the therapeutic agent in over an extended period of time (eg, when measured over at least about 6-12 months at room temperature or under accelerated storage conditions).

The therapeutic agents are preferably soluble in the pharmaceutical compositions of the present invention. The term soluble as associated with the therapeutic agents of the present invention refers to the ability of such therapeutic agents to form a homogeneous solution. The solubility of the therapeutic agent in the solution into which it is administered and transported to the target site of action (eg, brain cells and tissues) is preferably sufficient to ensure delivery of a therapeutically effective amount of the therapeutic agent to the target site of action. The solubility of therapeutic agents can be influenced by several factors. For example, significant factors that can influence protein solubility include ionic strength, amino acid sequence, and the presence of other co-solubilizing agents or salts (eg, calcium salts). In some embodiments, the pharmaceutical compositions are formulated such that calcium salts are not included in such compositions.

Suitable formulations in aqueous, pre-lyophilized, lyophilized or diluted form may contain the therapeutic agent of interest (eg, recombinant human arylsulfatase A) at various concentrations. In some embodiments, the formulations may contain the protein or therapeutic agent of interest at a concentration ranging from about 0.1 to 100 mg/mL (e.g., about 0.1 to 80 mg/mL, about 0.1 to 60 mg/ml, about 0.1 to 50 mg/ml, about 0.1 to 40 mg/ml, about 0.1 to 30 mg/ml, about 0.1 to 25 mg/ml, about 0.1 to 20 mg/ml, approximately 0.1 to 60 mg/ml, approximately 0.1 to 50 mg/ml, approximately 0.1 to 40 mg/ml, approximately 0.1 to 30 mg /ml, about 0.1 to 25 mg/ml, about 0.1 to 20 mg/ml, about 0.1 to 15 mg/ml, about 0.1 to 10 mg/ml, about 0 ,1 to 5 mg/ml, about 1 to 10 mg/ml, about 1 to 20 mg/ml, about 1 to 40 mg/ml, about 5 to 100 mg/ml, about 5 to 50 mg /ml or approximately 5 to 25 mg/ml). In some embodiments, the compositions of the present invention may contain a therapeutic agent at a concentration of at least or about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg/ml. In some embodiments, the formulations contain the therapeutic agent at a concentration of at least or about 25 mg/ml. In some embodiments, the formulations contain the therapeutic agent at a concentration of at least or about 30 mg/ml.

The compositions of the present invention are characterized by their portability in the form of aqueous solutions or in the form of diluted lyophilized solutions. As used herein, the terms tolerable and tolerable refer to the ability of the pharmaceutical compositions of the present invention not to cause an adverse reaction in a subject to whom such a composition is administered, or, alternatively, not to cause a serious adverse reaction in a subject to which such a composition is administered. In some embodiments, the pharmaceutical compositions of the present invention are well tolerated by the subject to whom such compositions are administered.

Many therapeutic agents, and in particular the proteins and enzymes of the present invention, require controlled pH and specific excipients to maintain their solubility and stability in the pharmaceutical compositions of the present invention. In table 3 below defines typical aspects of protein formulations that are considered to support the solubility and stability of the protein therapeutics of the present invention.

Table 3

Parameter Typical Range/Type Rationale for use pH 5-7.5 For stability Sometimes also for solubility Buffer type Acetate, succinate, citrate, histidine, phosphate or Tris To maintain optimal pH May also affect stability To maintain pH Buffer concentration 5-50 mM May also stabilize or add ionic strength To give Tonicity modifier NaCl, sugar, mannitol solutions isosmotic or isotonicity Surface active Polysorbate 20, Polysorbate 80 To stabilize the interfaces substance against shear Other Amino acids (for example, arginine) in concentrations from To increase solubility or P. DD IXO J1JD Γνΐ/ΐζχ /J, DD O zl -L Gch O J—> 44^9 several hundred mM stability

Buffers.

The pH of the formulation is an additional factor that can alter the solubility of the therapeutic agent (eg, enzyme or protein) in the aqueous formulation or in the formulation prior to lyophilization. Accordingly, the compositions of the present invention preferably contain one or more buffers. In some embodiments, the aqueous formulations contain an amount of buffer sufficient to maintain the optimum pH of the composition at about 4.0-8.0 (e.g., about 4.0, 4.5, 5.0, 5.5, 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.5 or 8.0). In some embodiments, the pH of the composition is about 5.0-7.5, about 5.5-7.0, about 6.0-7.0, about 5.5-6.0, about 5. 5-6.5, approximately 5.0-6.0, approximately 5.0-6.5 and approximately 6.0-7.5. Suitable buffers include, for example, acetate, citrate, histidine, phosphate, succinate, tris(hydroxymethyl)aminomethane (Tris) buffers and other organic acid buffers. In some embodiments, the buffer is phosphate.

The buffer concentration and pH range of the pharmaceutical compositions of the present invention are factors taken into account when adjusting or adjusting the tolerability of the formulation. In some embodiments, the buffering agent is present at a concentration ranging from about 1 mM to about 150 mM, or from about 10 mM to about 50 mM, or from about 15 mM to about 50 mM, or from about 20 mM to about 50 mM, or from about 25 mM to about 50 mM. In some embodiments, a suitable buffering agent is present at a concentration of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, or 150 mM.

In some embodiments, the buffer is present at a concentration not exceeding an upper limit of, for example, about 100, 90, 80, 60, 50, 40, 30, 20, 10, or 5 mM. In some embodiments, the buffer is present at a concentration not exceeding about 50 mM. In some embodiments, the buffer is present at a concentration not exceeding about 25 mM. In some embodiments, the buffer is present at a concentration not exceeding about 20 mM. In some embodiments, the buffer is present at a concentration not exceeding about 10 mM. In some embodiments, the buffer is present at a concentration not exceeding about 5 mM.

Tonicity.

In some embodiments, the aqueous, pre-lyophilized, lyophilized, or diluted form formulations contain an isotonicizing agent to maintain the isotonicity of the formulations. Generally, by isotonic it is meant that the compound of interest has essentially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of about 240 mOsm/kg to about 350 mOsm/kg. Isotonicity can be measured using, for example, vapor pressure or freezing point osmometers. Exemplary isotonicizing agents include, but are not limited to, glycine, sorbitol, mannitol, sodium chloride, and arginine. In some embodiments, suitable isotonicizing agents may be present in the aqueous and/or pre-lyophilization formulations at a concentration of about 0.01-5% (e.g., 0.05, 0.1, 0.15, 0.2 , 0.3, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0 or 5.0%) by weight. In some embodiments, lyophilization formulations contain an isotonicizing agent to maintain isotonicity of the formulations prior to lyophilization or diluted formulations.

Although isotonic solutions are generally preferred for parenteral administration of drugs, the use of isotonic solutions may alter the solubility for some therapeutic agents and, in particular, for some proteins and/or enzymes. Mildly hypertonic solutions (eg, up to 175 mM sodium chloride in 5 mM sodium phosphate at pH 7.0) and sugar-containing solutions (eg, up to 2% sucrose in 5 mM sodium phosphate at pH 7.0) have been demonstrated to be well tolerated. The most common approved CNS bolus formulation is saline (approximately 150 mM NaCl in water).

Stabilizers.

In some embodiments, the formulations may contain a stabilizer or lyoprotectant to protect the protein. Typically, a suitable stabilizer is a sugar, a non-reducing sugar and/or an amino acid. Exemplary sugars include, but are not limited to, dextran, lactose, mannitol, mannose, sorbitol, raffinose, sucrose and trehalose. Exemplary amino acids include, but are not limited to, arginine, glycine and methionine. Additional stabilizers may include sodium chloride, hydroxyethyl starch and polyvinylpyrrolidone. The amount of stabilizer in a lyophilized formulation is usually such that the formulation will be isotonic. However, hypertonic diluted formulations may also be suitable. In addition, the amount of stabilizer should not be so excessively low that an unacceptable amount of degradation/aggregation of the therapeutic agent occurs. Exemplary concentrations of stabilizer in the formulation may range from about 1 mM to about 400 mM (for example, from about 30 mM to about 300 mM and from about 50 mM to about 100 mM) or, alternatively, from 0. 1 to 15% (e.g., 1 to 10%, 5 to 15%, 5 to 10%) by weight. In some embodiments, the weight ratio of stabilizer to therapeutic agent is approximately 1:1. In other embodiments, the weight ratio of stabilizer to therapeutic agent may be approximately 0.1:1, 0.2:1, 0.25:1, 0.4:1, 0.5:1, 1:1, 2: 1, 2.6:1, 3:1, 4:1, 5:1, 10:1 or 20:1. In some embodiments suitable for lyophilization, the stabilizer is also a lyoprotectant.

In some embodiments, liquid compositions suitable for the present invention are

- 16 043909 contain amorphous substances. In some embodiments, liquid formulations suitable for the present invention contain significant amounts of amorphous materials (eg, sucrose-based formulations). In some embodiments, liquid formulations suitable for the present invention contain partially crystalline/partly amorphous substances.

Volume-forming agents.

In some embodiments, suitable lyophilization compositions may further comprise one or more bulking agents. A bulking agent is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake. For example, the bulking agent may improve the appearance of the lyophilized cake (eg, a substantially uniform lyophilized cake). Suitable bulking agents include, but are not limited to, sodium chloride, lactose, mannitol, glycine, sucrose, trehalose, hydroxyethyl starch. Exemplary concentrations of bulking agents range from about 1% to about 10% (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 , 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0%).

Surfactants.

In some embodiments, it is desirable to add a surfactant to the formulations. Exemplary surfactants include nonionic surfactants such as polysorbates (eg, polysorbates 20 or 80); poloxamers (eg poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl sarcosine; linoleyl-, myristyl- or cetylbetaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl betaine (eg lauroamidopropyl); myristamidopropyl, palmidopropyl or isostearamidopropyldimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; as well as the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and ethylene glycol-propylene glycol copolymers (e.g., Pluronics, PF68, etc.). Typically, the amount of surfactant added is such that it reduces protein aggregation and minimizes the formation of particulate matter or gas bubbles. For example, the surfactant may be present in the formulation at a concentration of about 0.001-0.5% (eg, about 0.005-0.05% or 0.005-0.01%). In particular, the surfactant may be present in the composition at a concentration of about 0.005, 0.01, 0.02, 0.1, 0.2, 0.3, 0.4 or 0.5%, etc. . Alternatively or in addition, the surfactant can be added to the lyophilized formulation, the pre-lyophilized formulation, and/or the reconstituted formulation.

Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the formulation (and/or in the lyophilized formulation and/or in the reconstituted formulation) provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to patients receiving them at the doses and concentrations used and include, without limitation, additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (for example, protein complexes with Zn); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium ion.

Formulations in aqueous, pre-lyophilized, lyophilized or reconstituted form according to the present invention can be evaluated based on analysis of product quality, reconstitution time (in the case of lyophilized form), reconstitution quality (in the case of lyophilized form), high molecular weight, moisture content and glass transition temperature. Typically, protein and product quality analysis involves analyzing the rate of protein degradation using methods including, but not limited to, size exclusion HPLC (SE-HPLC), cation exchange HPLC (CEX-HPLC), X-ray diffraction (XRD), modulated differential scanning calorimetry (mDSC), reverse phase HPLC (RP-HPLC), multi-angle light scattering (MALS), fluorescence analysis, ultraviolet absorption analysis, nephelometry, capillary electrophoresis (CE), SDS-PAGE and combinations thereof. In some embodiments, evaluating a product in accordance with the present invention may include the step of evaluating appearance (appearance of the liquid or cake).

The formulations (lyophilized or aqueous) can usually be stored for extended periods of time at room temperature. The storage temperature may typically be in the range of 0 to 45°C (eg, 4, 20, 25, 45°C, etc.). The compositions can be stored for periods ranging from several months to several years. Storage times are usually 24, 12, 6, 4.5, 3, 2 or 1 month. The compositions can be stored directly in the container used for administration, eliminating transfer steps.

The formulations can be stored directly in the lyophilization container (in the case of the lyophilized form), which can also function as a reconstitution vessel, excluding

- 17 043909 transfer stages. Alternatively, freeze-dried product formulations can be measured into smaller batches for storage. During storage, circumstances that cause protein degradation should generally be avoided, including but not limited to exposure to sunlight, UV radiation, other forms of electromagnetic radiation, overheating or hypothermia, rapid temperature shock, and mechanical shock.

Lyophilization.

The methods of the present invention can be used to lyophilize any materials, in particular therapeutic agents. Typically, the formulation prior to lyophilization further contains suitably selected excipients or other components, such as stabilizers, buffering agents, bulking agents, and surfactants, to prevent degradation of the compound of interest (e.g., aggregation, deamidation, and/or protein oxidation) , during freeze drying and storage. The lyophilization composition may contain one or more additional ingredients, including lyoprotectants or stabilizers, buffers, bulking agents, isotonicizing agents, and surfactants.

After mixing the substance of interest and any additional components with each other, the composition is lyophilized. Lyophilization usually involves three main steps: freezing, primary drying and secondary drying. Freezing is necessary to convert water into ice or some amorphous components of a composition into crystalline form. Primary drying is a process step in which ice is removed from a frozen product by direct sublimation at low pressure and temperature. Secondary drying is a process step in which bound water is removed from the product matrix using diffusion of residual water to the evaporation surface. The temperature of the product during secondary drying under normal conditions is higher than during primary drying. See Tang X. et al. (2004) Design of freeze-drying processes for Pharmaceuticals: Practical advice, Pharm. Res., 21:191-200; Nail S.L. et al. (2002) Fundamentals of freeze-drying, in Development and manufacture of protein pharmaceuticals. Nail S.L. editor New York: Kluwer Academic/Plenum Publishers, pp. 281-353; Wang et al. (2000) Lyophilization and development of solid proteins Pharmaceuticals, Int. J. Pharm., 203:1-60; Williams N.A. et al. (1984) The lyophilization of Pharmaceuticals; A literature review. J. Parenteral Sci. Technol., 38:48-59. Generally, any lyophilization process can be used within the scope of the present invention.

In some embodiments, an annealing step may be introduced during the initial freezing of the product. The annealing step can provide a reduction in overall cycle time. Without wishing to be bound by any theory, it is believed that the annealing step may help stimulate crystallization of the filler and the formation of larger ice crystals due to the recrystallization of small crystals formed during supercooling, which in turn improves dilution. Typically, the annealing step involves a temperature interval or temperature fluctuation during freezing. For example, the freezing temperature may be -40°C, and during the annealing step the temperature will be increased to, for example, -10°C, and this temperature will be maintained for a predetermined period of time. The duration of the annealing step can range from 0.5 to 8 hours (eg, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 6 and 8 hours). The annealing temperature can be between freezing temperature and 0°C.

Lyophilization can be carried out in a container such as a tube, bag, bottle, tray, vial (eg, glass vial), syringe, or any other suitable container. Containers can be disposable. Lyophilization can also be carried out on a large scale or on a small scale. In some cases, it may be desirable to lyophilize the protein formulation in the container in which the protein is to be reconstituted, in order to avoid a transfer step. The container in this case can, for example, be a bottle of 3, 4, 5, 10, 20, 50 or 100 cm ³ .

Many different freeze dryers are available for this purpose, such as the Hull commercial freeze dryer (SP Industries, USA), the Genesis laboratory freeze dryer (SP Industries), or any freeze dryer capable of adjusting the set parameters of the lyophilization process. Freeze drying is accomplished by freezing the composition and then sublimating ice from the frozen contents at a temperature suitable for primary drying. During initial freezing, the composition is brought to a temperature below about -20°C (e.g. -50, -45, -40, -35, -30, -25, etc.), typically in no more than about 4 hours (eg, no more than about 3 hours, no more than about 2.5 hours, no more than about 2 hours). Under this condition, the product temperature is typically lower than the eutectic point or collapse temperature of the composition. Typically, the shelf temperature during the primary drying phase will be in the range of approximately -30 to 25°C (assuming the product temperature remains below the melting point during primary drying) with a suitable pressure typically in the range of approximately 20- 250 mtorr. The time required for drying, which can range from several hours to several days, will be determined primarily by the composition, size and type of container in which the sample is contained (eg, a glass vial), as well as the volume of liquid.

- 18 043909

The secondary drying step is carried out at approximately 0-60°C depending primarily on the type and size of container used and the type of therapeutic protein. The time required for drying, which can range from several hours to several days, will then also be determined mainly by the volume of liquid.

As a general rule, lyophilization will result in a lyophilized formulation having a moisture content of less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, and less than about 0.5%.

Breeding.

Although the pharmaceutical compositions of the present invention are typically in aqueous form when administered to a subject, in some embodiments, the pharmaceutical compositions of the present invention are lyophilized. Such compositions must be reconstituted by adding one or more diluents before administration to a subject. At the appropriate stage, typically at an appropriate point in time before administration to the patient, the lyophilized formulation may be reconstituted with a diluent such that the concentration of protein in the reconstituted formulation is desired.

Various diluents can be used in accordance with the present invention. In some embodiments, a suitable diluent for dilution is water. Water used as a diluent can be treated in a number of ways, including reverse osmosis, distillation, deionization, filtration methods (eg, activated carbon filtration, microfiltration, nanofiltration) and combinations of these treatments. In general, the water should be suitable for injection, including, but not limited to, sterile water or bacteriostatic water for injection.

Additional exemplary diluents include pH buffered solution (eg, phosphate buffered saline), sterile saline, Elliott's solution, Ringer's solution, or dextrose solution. Suitable diluents may optionally contain a preservative. Exemplary preservatives include aromatic alcohols such as benzyl or phenyl alcohol. The amount of preservative used is determined by evaluating different preservative concentrations for protein compatibility and testing the effectiveness of the preservative. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it may be present in an amount of about 0.1-2.0%, about 0.5-1.5%, or about 1.0-1. 2%.

Diluents suitable for the present invention may contain a number of additives, including, but not limited to, pH buffers (eg, Tris, histidine), salts (eg, sodium chloride) and other additives (eg, sucrose), including those described above (for example, stabilizers, isotonic agents).

In accordance with the present invention, the lyophilized substance (for example, a protein such as recombinant human arylsulfatase A) can be diluted to a concentration of at least 25 mg/ml (for example, at least 50 mg/ml, at least 75 mg/ml , at least 100 mg/ml) and located in any range between these values. In some embodiments, the lyophilized substance (e.g., protein) can be reconstituted to a concentration ranging from about 1 to 100 mg/mL (e.g., about 1 to 50 mg/mL, 1 to 100 mg/mL, about 1 to about 5 mg/ml, about 1 to about 10 mg/ml, about 1 to about 25 mg/ml, about 1 to about 75 mg/ml, about 10 to about 30 mg/ml, about 10 to about 50 mg/ml, about 10 to about 75 mg/ml, about 10 to about 100 mg/ml, about 25 to about 50 mg/ml, about 25 to about 75 mg/ml, about 25 to about 100 mg/ml, about 50 to about 75 mg/ml, about 50 to about 100 mg/ml). In some embodiments, the protein concentration in the reconstituted formulation may be higher than the concentration in the formulation prior to lyophilization. In some embodiments, the lyophilized substance is diluted to a concentration of at least 25 mg/ml, such as 30 mg/ml. High concentrations of protein in the reconstituted formulation are considered particularly useful in cases where subcutaneous or intramuscular delivery of the reconstituted formulation is contemplated. In some embodiments, the protein concentration in the reconstituted formulation may be about 2-50 times (eg, about 2-20 times, about 2-10 times, or about 2-5 times) the concentration in the formulation prior to lyophilization. In some embodiments, the protein concentration in the reconstituted formulation may be at least about 2 times (eg, at least about 3, 4, 5, 10, 20, 40 times) the concentration in the formulation prior to lyophilization.

Dilution in accordance with the present invention can be carried out in any container. Exemplary containers suitable for the present invention include, but are not limited to, tubes, vials, syringes (e.g., single chamber or dual chamber),

- 19 043909 bags, bottles and trays. Suitable containers can be made from any materials such as glass, plastic, metal. Containers can be disposable or reusable. Breeding can also be done on a large scale or small scale.

In some cases, it may be desirable to lyophilize the protein formulation in the container in which the protein is to be reconstituted, in order to avoid a transfer step. The container in this case can, for example, be a bottle of 3, 4, 5, 10, 20, 50 or 100 cm ³ . In some embodiments, a suitable container for lyophilization and reconstitution is a dual-chamber syringe (eg, Lyo-Ject® syringes (Vetter)). For example, a two-chamber syringe may contain both lyophilized material and diluent, each of which is contained in a separate chamber separated by a seal (see Example 5). For reconstitution, a piston may be attached to the seal on the diluent side, which can be pressed to move the diluent into the product chamber so that the diluent can come into contact with the lyophilized substance and reconstitution can occur as described herein (see example 5).

The pharmaceutical compositions, formulations, and corresponding methods of the present invention are useful for delivering a variety of therapeutic agents to the CNS of a subject (eg, intrathecal, intraventricular, or intracisternal route) and for treating associated diseases. The pharmaceutical compositions of the present invention are particularly useful for delivering proteins and enzymes (eg, enzyme replacement therapy) to subjects suffering from lysosomal storage disorders. Lysosomal storage diseases are a group of relatively rare inherited metabolic disorders caused by defects in the functioning of lysosomes. Lysosomal diseases are characterized by the accumulation of uncleaved macromolecules in lysosomes, leading to an increase in the size and number of such lysosomes and ultimately to cellular dysfunction and clinical abnormalities.

Delivery to the central nervous system.

The various stable formulations described herein are believed to be generally suitable for delivering therapeutics to the CNS. The stable formulations of the present invention can be used for delivery to the CNS via a variety of techniques and routes, including, but not limited to, intraparenchymal, intracerebral, intraventricular intracerebral (ICV), intrathecal (eg, lumbar IT, cistern magna IT) administration, and any other techniques and routes of direct or indirect injection into the CNS and/or CSF. The term cisterna magna refers to the space around and below the cerebellum as a gap between the skull and the upper part of the spine. Typically, injection into the cisterna magna is also called cisterna magna delivery.

Intrathecal delivery.

In some embodiments, the replacement enzyme is delivered to the CNS in a formulation described herein. In some embodiments, the replacement enzyme is delivered to the CNS by administration into the cerebrospinal fluid (CSF) of the subject in need of treatment. In some embodiments, intrathecal administration is used to deliver the required replacement enzyme (eg, ASA protein) to the CSF. As used herein, intrathecal administration (also called intrathecal injection) refers to an injection into the spinal canal (the intrathecal space surrounding the spinal cord). Various techniques can be used, including but not limited to lumbar puncture. Exemplary methods are described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192, the contents of which are incorporated herein by reference.

In accordance with the present invention, the enzyme can be injected into any area surrounding the spinal canal. In some embodiments, the enzyme is injected into the lumbar region. As used herein, the term lumbar region or lumbar region refers to the area between the third and fourth lumbar vertebrae (in the lower back) and, more meaningfully, the L2-S1 region of the spine. Typically, intrathecal injection into the lumbar region or lumbar region is also called lumbar intrathecal delivery or lumbar intrathecal injection.

In some embodiments, therapeutic proteins, such as recombinant arylsulfatase A, are delivered by lumbar intrathecal administration, for example, delivered to the area between the third and fourth lumbar vertebrae (low back) and more specifically to the L2-S1 region of the spine. It is believed that lumbar intrathecal administration or delivery differs from delivery to the cisterna magna in that lumbar intrathecal administration or delivery in accordance with the present invention provides better and more efficient delivery to the distal spinal canal, whereas delivery to the cisterna magna, among other things, as generally does not provide good delivery to the distal spinal canal.

Intrathecal delivery device.

Various devices can be used for intrathecal delivery in accordance with the present invention. In some embodiments, the intrathecal administration device contains a fluid access port (eg, an injection port); a hollow body (eg, a catheter) having a first flow diaphragm in fluid communication with the fluid access port and a second flow diaphragm configured to be inserted into the spinal cord; and a locking mechanism for fixing the inserted hollow body in the spinal cord. By way of non-limiting example, a suitable locking mechanism includes one or more projections positioned on the surface of the hollow body and a suture ring mounted on the one or more projections to prevent the hollow body (eg, a catheter) from slipping out of the spinal cord. In various embodiments, the fluid access port includes a reservoir. In some embodiments, the fluid access port includes a mechanical pump (eg, an infusion pump). In some embodiments, the implanted catheter is connected to a reservoir (eg, for bolus delivery) or an infusion pump. The fluid access port can be implanted or external.

In some embodiments, intrathecal administration can be accomplished by lumbar puncture (ie, slow bolus) or via a port-catheter delivery system (ie, infusion or bolus). In some embodiments, the catheter is inserted between the lamina of the lumbar vertebrae and the tip is gently advanced into the intrathecal space to the desired level (usually L3-L4).

In some embodiments, intrathecal administration is accomplished through intermittent or continuous access to an implanted intrathecal drug delivery device (IDDD).

Compared to intravenous administration, the single dose volume suitable for intrathecal administration is generally small. Typically, intrathecal delivery in accordance with the present invention maintains a balance in CSF composition as well as the subject's intracranial pressure. In some embodiments, intrathecal delivery occurs in the absence of adequate removal of CSF from the subject. In some embodiments, a suitable single dose volume may be, for example, less than about 10, 8, 6, 5, 4, 3, 2, 1.5, 1 or 0.5 ml. In some embodiments, a suitable single dose volume may be approximately 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 1-3, 1-5, 1 .5-3, 1-4 or 0.5-1.5 ml. In some embodiments, intrathecal delivery in accordance with the present invention includes the step of initially removing the required amount of CSF. In some embodiments, less than about 10 ml (eg, less than about 9, 8, 7, 6, 5, 4, 3, 2, 1 ml) of CSF is initially removed before intrathecal administration. In these cases, a suitable single dose volume may be, for example, greater than about 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 ml.

Other devices for intrathecal administration of therapeutic compositions or formulations to an individual are described in US Pat. No. 6,217,552, incorporated herein by reference. Alternatively, the drug can be given intrathecally, for example through a single injection or continuous infusion. It should be understood that dosage treatment may occur in the form of a single dose or multiple doses.

For injection, the compositions of the present invention can be formulated as liquid solutions. In addition, the enzyme can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms are also included. The injection can be carried out, for example, as a bolus injection or a continuous infusion of the enzyme (eg, using infusion pumps).

Delivery methods other than intrathecal.

Therapeutic enzymes, such as recombinant arylsulfatase A, can be administered by routes other than intrathecal, such as by intracerebral injection into the lateral ventricles of a subject's brain. The injection can be carried out, for example, through a burr hole made in the subject's skull. In another embodiment, the enzyme and/or other pharmaceutical composition is administered through a surgically inserted shunt into a cerebral ventricle of a subject. For example, the injection can be carried out into the lateral ventricles, which are larger in size. In some embodiments, it is also possible to inject into the smaller third and fourth ventricles.

Alternatively, the pharmaceutical compositions can be administered by injection into the cistern magna or lumbar region of the subject.

Certain devices may be used to perform administration of the therapeutic composition. For example, formulations containing the necessary enzymes can be provided using an Ommaya reservoir, widely used for intrathecal administration of drugs for meningeal carcinomatosis (Lancet 2: 983-84, 1963). More specifically, in this method, a ventricular tube is inserted through an opening formed in the anterior horn of the spinal cord and connected to an Ommaya reservoir located under the skin of the scalp, and the reservoir is pierced under the skin for intrathecal delivery of a specific replacement enzyme introduced into the reservoir.

- 21 043909

Slow release/consistent delivery.

In another embodiment of the method of the present invention, the pharmaceutically acceptable formulation provides stable delivery, such as slow release, of an enzyme or other pharmaceutical composition used in the present invention to the body of a subject for at least one, two, three, four weeks or longer periods time after administration of the pharmaceutically acceptable composition to the subject.

As used herein, the term sustained delivery refers to the continuous delivery of the pharmaceutical composition of the present invention in vivo over a period of time after administration, preferably at least several days, a week, or several weeks. Sustained delivery of the composition may be demonstrated, for example, by a sustained therapeutic effect of the enzyme over time (eg, stable delivery of an enzyme may be demonstrated by a continued reduction in the number of storage granules in a subject). Alternatively, stable delivery of the enzyme can be demonstrated by detecting the presence of the enzyme in vivo over time.

Delivery to target tissues.

As discussed above, one of the surprising and important features of the present invention is that therapeutic agents, in particular enzyme replacements, administered using the inventive methods and compositions of the present invention are capable of efficient and widespread diffusion across the surface of the brain and to penetration into various layers or areas of the brain, including deep areas of the brain. In addition, the methods and compositions of the present invention provide effective delivery of therapeutic agents (eg, ASA enzyme) to various tissues, neurons or cells of the spinal cord, including the lumbar region, which is targeted by existing delivery methods in CNS such as ICV injection is difficult. In addition, the methods and compositions of the present invention provide delivery of sufficient amounts of therapeutic agents (eg, ASA enzyme) into the bloodstream and various peripheral organs and tissues.

Thus, in some embodiments, a therapeutic protein (eg, an ASA enzyme) is delivered to the central nervous system of a subject. In some embodiments, a therapeutic protein (eg, an ASA enzyme) is delivered to one or more target tissues of the brain, spinal cord, and/or peripheral organs. As used herein, the term target tissue refers to any tissue affected by a lysosomal storage disease to be treated, or any tissue that normally expresses a deficient lysosomal enzyme. In some embodiments, target tissues include those tissues that have detectable or abnormally high amounts of enzyme substrate, such as stored in cellular lysosomes of the tissue, in patients suffering from or susceptible to lysosomal storage disease. In some embodiments, target tissues include those tissues that exhibit a disease process, symptom, or sign associated with a disease. In some embodiments, target tissues include those tissues in which the deficient lysosomal enzyme is normally expressed at elevated levels. As used herein, the target tissue may be a brain target tissue, a spinal cord target tissue, and/or a peripheral target tissue. Exemplary target tissues are described in detail below.

Target brain tissues.

Generally, the brain can be divided into different regions, layers, and tissues. For example, meningeal tissue is a system of membranes covering the central nervous system, including the brain. The meninges contain three layers, including the dura mater, the arachnoid mater, and the pia mater. In general, the main function of the meninges and cerebrospinal fluid is to protect the central nervous system. In some embodiments, the therapeutic protein of the present invention is delivered to one or more layers of the meninges.

The brain has three main subdivisions, including the cerebrum, cerebellum, and brain stem. The cerebral hemispheres are located above most other brain structures and are covered by a cortical layer. Beneath the cerebrum lies the brainstem, which resembles a stalk to which the cerebrum is attached. At the back of the brain, below the cerebrum and behind the brain stem, is the cerebellum.

The diencephalon, located near the midline of the brain and above the midbrain, contains the thalamus, metathalamus, hypothalamus, epithalamus, prethalamus, and pretectal region. The midbrain, also called the mesencephalon, contains the roof, tegmentum, aqueduct and cerebral peduncles, the red nucleus and the nucleus of the third pair of cranial nerves. The midbrain is associated with vision, hearing, motor control, the sleep/wake cycle, concentration, and thermoregulation.

The tissue areas of the central nervous system, including the brain, can be characterized by the depth of the tissue. For example, tissues of the central nervous system (eg, brain) can be characterized as superficial or shallow tissues, intermediate tissues, and/or deep tissues.

In accordance with the present invention, a therapeutic protein (eg, a replacement enzyme) can be delivered to any appropriate target brain tissue(s) associated with the specific disease being treated in the subject. In some embodiments, a therapeutic protein (eg, a replacement enzyme) according to the present invention is delivered to a superficial or shallow target brain tissue. In some embodiments, the therapeutic protein of the present invention is delivered to the target mid-depth brain tissue. In some embodiments, the therapeutic protein of the present invention is delivered to deep target brain tissue. In some embodiments, the therapeutic protein of the present invention is delivered to a combination of superficial or shallow target brain tissue, intermediate target brain tissue, and/or deep target brain tissue. In some embodiments, the therapeutic protein of the present invention is delivered to deep brain tissue located at least 4, 5, 6, 7, 8, 9, 10 mm or more below the outer surface of the brain (or inward from her).

In some embodiments, therapeutic agents (eg, enzymes) are delivered to one or more superficial or shallow tissues of the brain. In some embodiments, the targeted superficial or shallow brain tissues are located within 4 mm of the surface of the brain. In some embodiments, the target superficial or shallow brain tissues are selected from pia mater tissue, cortical strip tissue, hippocampus, Virchow-Robin space, blood vessels in VR space, hippocampus, portions of the hypothalamus on the undersurface of the brain, optic nerves and tracts, the olfactory bulb and its projections and their combinations.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to one or more deep tissues of the brain. In some embodiments, the targeted superficial or shallow brain tissues are located 4 mm (e.g., 5, 6, 7, 8, 9, or 10 mm) below (or inward from) the surface of the brain. In some embodiments, the targeted deep brain tissues include the cortical band. In some embodiments, the target deep brain tissues include one or more of the diencephalon (e.g., hypothalamus, thalamus, prethalamus, subthalamus, etc.), hindbrain, lenticular nuclei, basal ganglia, caudate nucleus, putamen, amygdala body, globus pallidus and their combinations.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to one or more cerebellar tissues. In certain embodiments, the one or more target cerebellar tissues are selected from the group consisting of molecular layer tissues, Purkinje cell layer tissues, granular cell layer tissues, cerebellar peduncles, and combinations thereof. In some embodiments, therapeutic agents (e.g., enzymes) are delivered to one or more deep cerebellar tissues, including, but not limited to, Purkinje cell layer tissue, granular cell layer tissue, deep cerebellar white matter tissue (e.g., deep versus granular layer cells) and deep tissue of the cerebellar nuclei.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to one or more brain stem tissues. In some embodiments, the one or more target brainstem tissues include brainstem white matter tissue and/or brainstem nuclear tissue.

In some embodiments, therapeutic agents (e.g., enzymes) are delivered to various tissues of the brain, including, but not limited to, gray matter, white matter, periventricular zones, pia and arachnoid maters, meninges, neocortex, cerebellum, deep tissues in cerebral cortex, molecular layer, caudate/putamen region, midbrain, deep regions of the pons or spinal cord, and combinations thereof.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to various cells in the brain, including, but not limited to, neurons, glial cells, perivascular cells, and/or meningeal cells. In some embodiments, the therapeutic protein is delivered to deep white matter oligodendrocytes.

Spinal cord.

Typically, areas or tissues of the spinal cord can be characterized by the depth of the tissue. For example, spinal cord tissues can be characterized as superficial or shallow tissues, intermediate tissues, and/or deep tissues.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to one or more superficial or shallow tissues of the spinal cord. In some variants

- 23 043909 implementation of the target superficial or shallow tissue of the spinal cord is located within 4 mm from the surface of the spinal cord. In some embodiments, the targeted superficial or shallow spinal cord tissue comprises pia mater and/or white matter tracts.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to one or more deep tissues of the spinal cord. In some embodiments, the target deep spinal cord tissue is located within 4 mm inward of the surface of the spinal cord. In some embodiments, the target deep tissue of the spinal cord includes gray matter and/or ependymal cells of the spinal cord.

In some embodiments, therapeutic agents (eg, enzymes) are delivered to spinal cord neurons.

Peripheral target tissues.

As used herein, peripheral organs or tissues refer to any organs or tissues that are not part of the central nervous system (CNS). Peripheral target tissues may include, but are not limited to, the blood system, liver, kidney, heart, endothelium, bone marrow and bone marrow-derived cells, spleen, lung, lymph node, bone, cartilage, ovary and testis. In some embodiments, a therapeutic protein (eg, a replacement enzyme) in accordance with the present invention is delivered to one or more peripheral target tissues.

Biodistribution and bioavailability.

In various embodiments, once delivered to the target tissue, the therapeutic agent (eg, ASA enzyme) is localized intracellularly. For example, a therapeutic agent (eg, an enzyme) may be localized to exons, axons, lysosomes, mitochondria, or vacuoles of target cells (eg, neurons such as Purkinje cells). For example, in some embodiments, intrathecally administered enzymes exhibit translocation dynamics in which the enzyme moves in the perivascular space (eg, through pulsatile convective mechanisms). In addition, active axonal transport mechanisms associated with the association of the administered protein or enzyme with neurofilaments may also contribute to or otherwise contribute to the distribution of intrathecally administered proteins or enzymes into deeper tissues of the central nervous system.

In some embodiments, a therapeutic agent (eg, an ASA enzyme) delivered in accordance with the present invention can achieve therapeutically or clinically effective levels or activities in various target tissues described herein. As used herein, a therapeutically or clinically effective level or activity is a level or activity sufficient to provide a therapeutic effect in the target tissue. The therapeutic effect may be objective (i.e., measured by a specific test or marker) or subjective (i.e., the subject indicates or perceives the effect). For example, a therapeutically or clinically effective level or activity may be a level or activity of an enzyme sufficient to reduce the severity of symptoms associated with a disease in the target tissue (eg, GAG accumulation).

In some embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention may achieve an enzyme level or activity of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 95% of the normal level or activity of the corresponding lysosomal enzyme in the target tissue. In some embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention may achieve an enzyme level or activity increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the control or baseline (eg, endogenous levels or activities in the absence of treatment). In some embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention can achieve increased enzyme levels or activity of at least about 10, 20, 40, 50, 60, 70, 80, 90, 100 , 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 nmol/h/mg in the target tissue.

In some embodiments, the methods of the present invention are particularly useful for targeting the lumbar region. In some embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention can achieve increased levels or activity of the enzyme in the lumbar region of at least about 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nmol/h/mg.

Typically, therapeutic agents (eg, enzyme replacements) delivered in accordance with the present invention have a fairly long half-life from the CSF and target tissues of the brain, spinal cord and peripheral organs. In some embodiments, the therapeutic agent (eg, enzyme replacement) delivered in accordance with the present invention may have a half-life of at least

- 24 043909 approximately 30, 45, 60, 90 min, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18, 20, 25, 30, 35, 40 h, up to 3 , up to 7, up to 14, up to 21 days or up to a month. In some embodiments, in some embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention may maintain detectable levels or activity in the CSF or bloodstream after 12, 24, 30, 36, 42, 48, 54. 60, 66, 72, 78, 84, 90, 96, 102 hours or a week after administration. The detectable level or activity can be determined using various methods known in the art.

In certain embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention reaches a concentration of at least 30 μg/mL in the tissues and cells of the subject's CNS after administration (e.g., one week, 3 days, 48 , 36, 24, 18, 12, 8, 6, 4, 3, 2, 1 hour, 30 minutes or less after intrathecal administration of the pharmaceutical composition to the subject). In certain embodiments, the therapeutic agent (e.g., enzyme replacement) delivered in accordance with the present invention reaches a concentration of at least 20 μg/ml, at least 15 μg/ml, at least 10 μg/ml, at least at least 7.5 µg/ml, at least 5 µg/ml, at least 2.5 µg/ml, at least 1.0 µg/ml or at least 0.5 µg/ml, in target tissues or cells of a subject (e.g., in brain tissue or neurons) following administration to such subject (e.g., one week, 3 days, 48, 36, 24, 18, 12, 8, 6, 4, 3, 2, 1 hour, 30 minutes or less following intrathecal administration of such pharmaceutical compositions to a subject).

Treatment of the disease metachromatic leukodystrophy (MLD).

Metachromatic leukodystrophy (MLD), also known as MLD syndrome, is an autosomal recessive disorder caused by deficiency of the enzyme arylsulfatase A (ASA). ASA, encoded by the ARSA gene in humans, is an enzyme that degrades cerebroside-3-sulfate or the sphingolipid 3-0-sulfogalactosylceramide (sulfatide) into cerebroside and sulfate. In the absence of the enzyme, sulfatides accumulate in the nervous system (eg, myelin sheaths, neurons, and glial cells) and, to a lesser extent, in visceral organs. The consequence of these molecular and cellular events is progressive demyelination and axonal loss in the CNS and PNS, which is accompanied by clinical manifestations of severe motor and cognitive dysfunction.

The defining clinical feature of this disorder is degeneration of the central nervous system (CNS), which leads to cognitive impairment (eg, mental retardation, nervous disorders, and blindness, among others).

MLD can occur in young children (late infantile form), with affected children typically beginning to show symptoms soon after the first year of life (eg, around 15–24 months of age), and children usually do not live beyond 5 years . MLD can manifest in children (juvenile form), with affected children typically demonstrating cognitive impairment by approximately 3–10 years of age, and life expectancy may vary (eg, in the range of 10–15 years after symptom onset). MLD can manifest in adults (the adult-onset form) and can appear in individuals at any age (eg, typically 16 years of age or later). The progression of the disease can vary significantly.

The compositions and methods of the present invention can be used to effectively treat individuals suffering from or susceptible to MLD. Certain methods of the present invention for the treatment of MLD typically include the step of intrathecal administration to a subject in need of treatment of a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and at an interval between administrations for a period of treatment sufficient to observe at least one indicator of treatment effectiveness. In accordance with many of the methods provided in the present invention, the subject does not experience serious adverse effects associated with the administration of recombinant arylsulfatase A.

The terms treat or cure as used herein refer to reducing the intensity of one or more symptoms associated with a disease, preventing or delaying the onset of one or more symptoms of a disease, reducing the severity or frequency of one or more symptoms of a disease, and/or slowing, stabilizing or reducing the rate of progression of deterioration in one or more functions associated with the disease. Exemplary symptoms include, but are not limited to, intracranial pressure, ex vacuo hydrocephalus, accumulation of sulfated glycolipids in myelin sheaths in the central and peripheral nervous systems and visceral organs, progressive demyelination and axonal loss in the CNS and PNS, and/or motor and cognitive dysfunction. .

In some embodiments, treatment relates to partial or complete alleviation, reduction in intensity, attenuation, inhibition, delay in onset of manifestation, reduction in severity and/or incidence, slowing of progression, stabilization, or elimination of neurological impairment in a patient with MLD. As used herein, the term neurological disorder includes various symptoms associated with impaired functioning.

- 25 043909 central nervous system (eg brain and spinal cord). In some embodiments, various symptoms of MLD are associated with dysfunction of the peripheral nervous system (PNS).

Motor functions.

In some embodiments, the neurological impairment in a patient with MLD is characterized by deterioration in motor function, such as gross motor function. In some embodiments, treatment effectiveness includes a measure that is associated with one or more motor functions.

In some embodiments, methods for treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and at an interval between administrations for a period of treatment sufficient to effect improvement. stabilization or reduction in the rate of deterioration of one or more motor functions compared to the initial level. In some embodiments, administration of the recombinant ASA enzyme further results in improvement, stabilization, or reduction in the rate of decline of one or more cognitive, adaptive, and/or executive functions.

One or more motor functions may include, for example, a gross motor function. It will be understood that gross motor function can be assessed using any appropriate method. For example, in some embodiments, gross motor function is assessed by the change in motor function from baseline using total pretest scores or percentages on a test of gross motor functioning, such as the Test of Gross Motor Functioning-88 (GMFM-88) or the Test of Gross Motor Functioning. motor skills-66 (GMFM-66). In some embodiments, the subject being treated has a baseline GMFM-88 score greater than 40%. In some embodiments, the subject being treated has a baseline GMFM-88 score of less than 40%.

In some embodiments, administration of a recombinant ASA enzyme in accordance with the methods disclosed herein results in less impairment in motor function than the impairment that would typically be observed without administration. For example, in some embodiments, administration of recombinant ASA enzyme in accordance with the methods of the present invention results in a reduction in the GMFM-88 score of less than 10, 20, 30, 40, or 50%.

In some embodiments, administration of a recombinant ASA enzyme in accordance with the methods disclosed herein results in significant stabilization of motor function, such as significant stabilization of a score, such as a GMFM-88 score. By significant stabilization is meant that there is no deterioration or worsening for a period of time, such as during a period of treatment and/or during a period that would normally be expected to deteriorate or worsen in the absence of treatment.

In some embodiments, administration of a recombinant ASA enzyme in accordance with the methods disclosed herein results in an improvement in motor function, such as an improvement in a score, such as a GMFM-88 score.

Biomarkers.

In certain embodiments, treatment effectiveness involves change(s) in the level(s) of one or more biomarkers, such as those that accumulate or are present at reduced levels in patients with MLD.

In some embodiments, methods of treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and at some interval between administrations for a period of treatment sufficient to reduce levels biomarker accumulating in MLD compared with baseline biomarker levels.

The biomarker that accumulates in MLD may be, for example, sulfatide, lysosulfatide, or both. In some embodiments, the biomarker is a sulfatide. The biomarker may be a biomarker that accumulates in one or more body tissues and/or one or more body fluids in patients with MLD. For example, in some embodiments, the biomarker accumulates in a physiological fluid selected from the group consisting of cerebrospinal fluid, urine, blood, and serum. In some embodiments, the physiological fluid is cerebrospinal fluid.

In certain embodiments, the baseline level of sulfatides in the cerebrospinal fluid of the subject being treated is greater than a certain level, such as greater than about 0.1 μg/ml, greater than about 0.2 μg/ml, or greater than about 0.3 μg/ml. In some embodiments, administration of the recombinant ASA enzyme in accordance with the methods disclosed herein results in a reduction in cerebrospinal fluid sulfatide levels, e.g., greater than about 0.1 μg/mL or greater than about 0.2 μg/mL .

- 26 043909

In some embodiments, treatment results in reduced accumulation of sulfatides in various tissues or body fluids. In some embodiments, treatment results in reduced accumulation of sulfatides in target brain tissues, spinal cord neurons, and/or peripheral target tissues. In certain embodiments, sulfatide accumulation is reduced by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more compared to control or baseline. In some embodiments, sulfatide accumulation is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to control or baseline. It will be understood that sulfatide accumulation can be assessed using any suitable method. For example, in some embodiments, sulfatide accumulation is measured by Alcian blue staining. In some embodiments, sulfatide accumulation is measured by staining with LAMP-1.

In some embodiments, methods of treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose and at some interval between administrations for a period of treatment sufficient to increase levels biomarkers reduced in MLD in brain tissue compared to baseline biomarker levels.

The brain tissue can be any tissue from the brain of patients with MLD in which biomarker levels are typically reduced, such as the deep white matter of the brain. Biomarkers that may be useful include certain metabolites such as N-acetylaspartate. Levels of N-acetyl aspartate in brain tissue can be assessed, for example, using proton magnetic resonance spectroscopy.

Involvement in brain damage.

In certain embodiments, the effectiveness of treatment is provided by an indicator related to involvement of brain damage. In certain embodiments, methods for treating metachromatic leukodystrophy (MLD) syndrome are provided, comprising the step of intrathecally administering to a subject in need of treatment a recombinant arylsulfatase A enzyme at a therapeutically effective dose and at an interval between administrations for a period of treatment sufficient to stabilize or reduce the degree of involvement. in brain damage compared to baseline.

Involvement in brain damage can be assessed, for example, using non-invasive methods, such as imaging techniques. Suitable assessment methods include qualitative, quantitative and semi-quantitative methods. For example, a non-invasive imaging method for assessing brain damage involvement in patients with MLD is an MRI (magnetic resonance imaging) MLD severity score. (See, for example, Eichler et al. AJNR Am. J. Neuroradiol. 2009 Nov; 30(10):1893-7, the entire contents of which are incorporated herein by reference.) In some embodiments, administration of the recombinant ASA enzyme results in a decrease in the subject's MLD severity score, as determined by MRI, compared to baseline. In some embodiments, administration of the recombinant ASA enzyme results in stabilization of the subject's MLD severity score, as determined by MRI, compared to baseline.

Additional indicators of treatment effectiveness.

The methods of the present invention alternatively or additionally lead to one or more of the other treatment efficacy measures discussed herein. In some embodiments, treatment results in reduced vacuolation of neurons (eg, Purkinje cell neurons). In certain embodiments, neuronal vacuolation is reduced by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more compared to control or baseline. In some embodiments, vacuolation is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to control or baseline.

In some embodiments, treatment results in increased ASA enzyme activity in various tissues. In some embodiments, treatment results in increased ASA enzyme activity in target brain tissues, spinal cord neurons, and/or peripheral target tissues. In some embodiments, ASA enzyme activity is increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 1000% or more compared to control or baseline. In some embodiments, ASA enzyme activity is increased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to control or baseline. In some embodiments, the increased ASA enzymatic activity is at least about 10, 20, 40, 50, 60, 70, 80, 90, 100, 15θ, 200, 250, 300, 350, 400, 450, 500, 550, 600 nmol/h/mg or more. In some embodiments, the enzymatic activity of ASA is increased by

- 27 043909 lumbar region. In some embodiments, increased enzymatic activity

ASA in the lumbar region is at least about 2000, 3000, 4000, 5000, 6000, 7000,

8000, 9000, 10000 nmol/h/mg or more.

In some embodiments, treatment results in reduced progression of cognitive loss. In certain embodiments, the progression of cognitive loss is reduced by approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 % or more compared to control or baseline. In some embodiments, the treatment relates to reduced developmental delay. In certain embodiments, developmental delay is reduced by approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more compared to control or baseline.

In some embodiments, treatment relates to increased survival (eg, survival time). For example, treatment may result in an increased life expectancy for the patient. In some embodiments, treatment in accordance with the present invention results in a patient's life expectancy increased by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 100% , approximately 105%, approximately 110%, approximately 115%, approximately 120%, approximately 125%, approximately 130%, approximately 135%, approximately 140%, approximately 145%, approximately 150%, approximately 155%, approximately 160%, approximately 165%, approximately 170%, approximately 175%, approximately 180%, approximately 185%, approximately 190%, approximately 195%, approximately 200% or greater compared to the average life expectancy of one or more control individuals with a similar disease without treatment. In some embodiments, treatment in accordance with the present invention results in a patient's life expectancy increased by more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years or more compared with the average life expectancy of one or more control individuals with similar disease without treatment. In some embodiments, treatment in accordance with the present invention results in long-term survival of the patient. As used herein, the term long-term survival refers to a survival time or life expectancy of more than about 40, 45, 50, 55, 60 years or more.

The terms improve, increase or decrease as used herein indicate values compared to control. In some embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual before initiation of a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein . A control individual is an individual affected by the same form of MLD (eg, late infantile, juvenile, or adult form) who is approximately the same age and/or sex as the treated individual (to ensure that the stages of the disease are treatment individual and control individual(s) are matched).

Subjects.

An individual (also referred to as a patient or subject) being treated is an individual (eg, fetus, infant, child, adolescent, or adult) who has MLD or is likely to develop MLD (ie, is at risk of developing MLD).

In many embodiments, the subject is a mammal, such as a human.

In some embodiments, the subject's age is sixteen years or less, such as twelve years or less, nine years or less, six years or less, four years or less, three years or less, two years or less, 18 months or less, 12 months or less or 6 months or less.

Subjects may have any of the forms of MLD, such as the adult form, the juvenile form, or the late infantile form.

Subjects may or may not exhibit at least one symptom of MLD discussed herein at the time of initiation of treatment.

Subjects may or may not have been diagnosed with MLD at the time of treatment initiation. For example, in some embodiments, the subject has not been identified as being at risk for developing MLD.

- 28 043909

An individual may have residual endogenous ASA expression and/or activity or no measurable activity. For example, an individual having MLD may have ASA expression levels of less than about 30-50%, less than about 25-30%, less than about 20-25%, less than about 15-20%, less than about 10-15%, less than about 5-10%, less than about 0.1-5% of normal ASA expression levels.

In some embodiments, the individual is an individual who has recently been diagnosed with a disease. In general, early treatment (treatment that is started as soon as possible after diagnosis) is important to minimize the effects of the disease and maximize the beneficial effects of treatment.

Immune tolerance.

Typically, intrathecal administration of a therapeutic agent (eg, enzyme replacement) in accordance with the present invention does not result in severe adverse effects in the subject. As used herein, severe adverse effects include, but are not limited to, significant immune response, toxicity, or death. As used herein, the term significant immune response refers to strong or severe immune responses, such as adaptive T-cell immune responses.

Thus, in many embodiments, the methods of the present invention do not provide for concomitant immunosuppressive therapy (ie, any immunosuppressive therapy administered as pretreatment/preconditioning or in parallel to the method). In some embodiments, the methods of the present invention do not involve inducing immune tolerance in the subject being treated. In some embodiments, the methods of the present invention do not involve pre-treating or preconditioning the subject with a T-cell immunosuppressive agent.

In some embodiments, intrathecal administration of therapeutic agents may provide an immune response to the agents. Thus, in some embodiments, it may be useful to ensure that a subject receiving enzyme replacement is tolerant to enzyme replacement therapy. Immune tolerance can be induced using various methods known in the art. For example, an initial regimen of a T-cell immunosuppressive agent such as cyclosporine A (CsA) and an antiproliferative agent such as azathioprine (Aza) for 30 to 60 days may be used in combination with weekly low-dose intrathecal infusions of the required replacement enzyme.

Any immunosuppressive agent known to one skilled in the art can be used in conjunction with the combination therapy of the present invention. Such immunosuppressive agents include, but are not limited to, cyclosporine, FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such as etanercept (see, e.g., Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; Nevins , 2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J. Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226; Potter et al. ., 1999, Ann. N.Y. Acad. Sci. 875, 159174; Slavik et al., 1999, Immunol. Res. 19, 1-24; Gaziev et al., 1999, Bone Marrow Transplant. 25, 689-696; Henry , 1999, Clin. Transplant. 13, 209-220; Gummert et al., 1999, J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al., 2000, Transplantation 69, 1275-1283). The anti-IL2 receptor (alpha subunit) antibody daclizumab (eg, Zenapax.TM.), which has demonstrated efficacy in transplant patients, can also be used as an immunosuppressive agent (see, e.g., Wiseman et al., 1999, Drugs 58 , 1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619; Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard et al., 1999, Pharmacotherapy 19, 1127-1137; Eckhoff et al., 2000, Transplantation 69, 1867-1872; Ekberg et al., 2000, Transpl. Int. 13, 151-159). Additional immunosuppressive agents include, but are not limited to, anti-CD2 (Branco et al., 1999, Transplantation 68, 1588-1596; Przepiorka et al., 1998, Blood 92, 4066-4071), anti-CD4 (Marinova Mutafchieva et al., 2000, Arthritis Rheum. 43, 638-644; Fishwild et al., 1999, Clin. Immunol. 92, 138-152) and CD40 ligand antibody (Hong et al., 2000, Semin. Nephrol. 20, 108-125 ; Chirmule et al., 2000, J. Virol. 74, 3345-3352; Ito et al., 2000, J. Immunol. 164, 1230-1235).

Introduction.

The inventive methods of the present invention include single as well as multiple administrations of a therapeutically effective amount of the therapeutic agents (eg, enzyme replacement) described herein. Therapeutics (eg, enzyme replacement) may be administered at regular intervals depending on the nature, severity, and severity of the condition (eg, lysosomal storage disease) in the subject. In some embodiments, a therapeutically effective amount of the therapeutic agents (e.g., enzyme replacements) of the present invention may be administered intrathecally periodically at regular intervals (e.g., once a year, once every six months,

- 29 043909 once every five months, once every three months, every two months (once every two months), monthly (once a month), every two weeks (once every two weeks), weekly).

In some embodiments, intrathecal administration may be used in combination with other routes of administration (eg, intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (eg, oral or nasal)). In some embodiments, administration by such other routes (e.g., intravenous administration) may be administered no more frequently than every two weeks, monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once a year.

As used herein, the term therapeutically effective amount is broadly defined as the total amount of therapeutic agent contained in the pharmaceutical composition of the present invention. Typically, the therapeutically effective amount is sufficient to achieve a significant beneficial effect in a subject (eg, treating, modulating, curing, preventing and/or ameliorating the underlying disease or condition). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect, such as an amount sufficient to modulate lysosomal enzyme receptors or their activity, thereby providing treatment for such lysosomal storage disease or symptoms thereof (e.g. , reduction in the number of striatal bodies or elimination of their presence or frequency of their appearance or cellular vacuolation after administration of the compositions of the present invention to the subject). Typically, the amount of therapeutic agent (eg, recombinant lysosomal enzyme) administered to a subject in need thereof will depend on the characteristics of the subject. Such characteristics include the condition, severity of the disease, general health, age, sex and body weight of the subject. One of ordinary skill in the art will readily be able to determine appropriate dosages depending on these and other interrelated factors. In addition, both objective and subjective assays may optionally be used to identify optimal dose ranges.

A therapeutically effective amount is typically administered according to a dosage regimen, which may include multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and/or the appropriate unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration, combination with other pharmaceutical agents. In addition, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical used; the specific composition used; age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the particular fusion protein used; duration of treatment and similar factors well known in the medical field.

In some embodiments, the therapeutically effective dose is or greater than about 1.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg. In specific embodiments, the therapeutically effective dose is or greater than about 10 mg. In specific embodiments, the therapeutically effective dose is or greater than about 30 mg. In specific embodiments, the therapeutically effective dose is or greater than about 100 mg. In some embodiments, the therapeutically effective dose is less than about 500, 450, 400, 350, 300, 250, or 200 mg. In specific embodiments, the therapeutically effective dose is less than about 200 mg. In some embodiments, the therapeutically effective dose is in the range of about 1-100 mg, about 5-100 mg, about 10-90 mg, about 10-80 mg, about 5-70 mg, about 5-60 mg, about 5-60 mg, about 10-100 mg, about 10-90 mg, about 10-80 mg, about 10-70 mg, about 10-60 mg or about 10-50 mg. In some embodiments, the therapeutically effective dose is in the range of from about 100 mg to about 200 mg.

In some embodiments, the therapeutically effective dose is in the range of from about 0.005 mg/kg brain weight to 500 mg/kg brain weight, such as from about 0.005 mg/kg brain weight to 400 mg/kg brain weight, from about 0.005 mg/kg brain weight to 300 mg/kg brain weight, from about 0.005 mg/kg brain weight to 200 mg/kg brain weight, from about 0.005 mg/kg brain weight to 100 mg/kg brain weight , from about 0.005 mg/kg brain weight to 90 mg/kg brain weight, from about 0.005 mg/kg brain weight to 80 mg/kg brain weight, from about 0.005 mg/kg brain weight to 70 mg/ kg brain weight, from about 0.005 mg/kg brain weight to 60 mg/kg brain weight, from about 0.005 mg/kg brain weight to 50 mg/kg brain weight, from about 0.005 mg/kg brain weight up to 40 mg/kg brain weight, from approximately 0.005 mg/kg brain weight to 30 mg/kg brain weight, from approximately 0.005 mg/kg brain weight to 25 mg/kg brain weight, from approximately 0.005 mg/kg brain weight to 20 mg/kg brain weight, from approximately

0.005 mg/kg brain weight to 15 mg/kg brain weight, from approximately 0.005 mg/kg brain weight to 10 mg/kg brain weight.

In some embodiments, the therapeutically effective dose is greater than about 0.1 mg/kg brain weight, greater than about 0.5 mg/kg brain weight, greater than about 1.0 mg/kg brain weight, greater than about 3 mg/kg brain weight brain, exceeds approximately 5 mg/kg brain weight, exceeds approximately 10 mg/kg brain weight, exceeds approximately 15 mg/kg brain weight, exceeds approximately 20 mg/kg brain weight, exceeds approximately 30 mg/kg brain weight brain, exceeds approximately 40 mg/kg brain weight, exceeds approximately 50 mg/kg brain weight, exceeds approximately 60 mg/kg brain weight, exceeds approximately 70 mg/kg brain weight, exceeds approximately 80 mg/kg brain weight brain, exceeds approximately 90 mg/kg brain weight, exceeds approximately 100 mg/kg brain weight, exceeds approximately 150 mg/kg brain weight, exceeds approximately 200 mg/kg brain weight, exceeds approximately 250 mg/kg brain weight brain, exceeds approximately 300 mg/kg brain weight, exceeds approximately 350 mg/kg brain weight, exceeds approximately 400 mg/kg brain weight, exceeds approximately 450 mg/kg brain weight, exceeds approximately 500 mg/kg brain weight brain.

In some embodiments, the therapeutically effective dose may also be defined in mg/kg body weight. As one skilled in the art will appreciate, brain weight values and body weight values can be correlated. Dekaban AS. Changes in brain weights during the span of human life: relation of brain weights to body heights and body weights, Ann Neurol 1978; 4:345-56. Thus, in some embodiments, the doses can be converted, as shown in table. 4.

Table 4

Correlation between weight values brain, values body weight: Age and age of male persons (years) Head weight gender brain (kg) Weight body (kg)

(31-43 months) 1.27 15.55

4-5 1.30 19.46

In some embodiments, the therapeutically effective dose may also be defined in mg/15 cc. see CSF. As will be appreciated by one skilled in the art, therapeutically effective doses in terms of brain weight values and body weight values can be converted to mg/15 cc. see CSF. For example, CSF volume in adults is approximately 150 mL (Johanson CE, et al. Multiplicity of cerebrospinal fluid functions: New challenges in health and disease, Cerebrospinal Fluid Res. 2008 May 14;5:10). Thus, injections in adults of a single dose of 0.1 to 50 mg of protein would result in doses of approximately 0.01 mg/15 ^cc . CSF (0.1 mg) to 5.0 mg/15 cm ³ . CSF (50 mg) for adults.

It should also be understood that for any particular subject, specific dosage regimens must be adjusted over time according to individual need and the professional judgment of the practitioner administering or supervising the administration of enzyme replacement therapy, and that the dosage ranges set forth herein are provided as a guide only. example and do not imply a limitation on the scope or practical implementation of the claimed invention.

In certain embodiments, the recombinant arylsulfatase A enzyme is administered at some interval between administrations, such as at an equal interval. For example, the interval between administrations may be a week, two weeks or a month.

In certain embodiments, the recombinant arylsulfatase A enzyme is administered during the treatment period. The treatment period may be predetermined, or it may be adjusted depending on the patient's response to treatment, including without limitation possible adverse symptoms and/or evidence indicating the effectiveness of treatment, as discussed herein. For example, the treatment period may be at least 6 months, at least 9 months, at least 12 months, at least 24 months, or even longer.

It is believed that in some embodiments, subjects will be treated for a certain period of treatment, provided with a certain period during which they do not receive treatment or receive alternative treatment, and then again provided with another period of treatment.

Sets.

The present invention further provides kits or other products that contain the composition of the present invention and provide instructions for its reconstitution (in the case of a lyophilized form) and/or use. Kits or other articles may include a container, IDDD, catheter, and any other articles, devices, or equipment useful in intrathecal administration and associated surgery. Suitable containers include, for example, bottles, vials, syringes (eg pre-filled syringes), ampoules, cartridges, reservoirs or lyo-ject syringes. The container can be made from a number of materials, such as glass or plastic. In some embodiments, the container is a pre-filled syringe. Suitable prefilled syringes include, but are not limited to, borosilicate glass syringes coated with silicone by sintering, borosilicate glass syringes coated with silicone by spraying, or plastic polymer syringes containing no silicone.

Typically, the container may contain the formulations and a label located on or associated with the surface of the container, which may provide directions for reconstitution and/or use. For example, the label may indicate that the formulation is diluted to the protein concentrations described above. The label may further indicate that the composition is useful or intended, for example, for IT administration. In some embodiments, the container may contain a single dose of a stable formulation containing a therapeutic agent (eg, enzyme replacement). In various embodiments, a single dose of a stable formulation is present in a volume of less than about 15, 10, 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1. 0 or 0.5 ml. Alternatively, the container containing the formulation may be a reusable vial that allows for repeated administrations (eg, 2-6 administrations) of the formulation. Kits or other articles may further include a second container containing a suitable diluent (eg, BWFI, saline, buffered saline). When mixing diluent and formulation, the final concentration of protein in the diluted formulation will typically be at least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 25 mg/ml, at least at least 50 mg/ml, at least 75 mg/ml, at least 100 mg/ml). In some embodiments, the final protein concentration is at least 25 mg/ml, such as 30 mg/ml. Kits or other products may further include other materials that are commercially and consumer appropriate, including other buffers, diluents, filters, needles, IDDDs, catheters, syringes, and package inserts.

A more complete understanding of the present invention will be achieved by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. Although certain compounds, compositions and methods described herein have been specifically described in accordance with certain embodiments, the following examples serve only as illustrations of the compounds of the present invention and are not intended to be limiting. All references cited are incorporated by reference.

Examples

Example 1: Intrathecal administration of recombinant arylsulfatase A is safe and relieves symptoms in human patients with late infantile and juvenile metachromatic leukodystrophy.

Clinical forms of metachromatic leukodystrophy are generally subdivided based on age of onset, with late infantile metachromatic leukodystrophy associated with onset at three years of age or less, juvenile metachromatic leukodystrophy associated with onset between four and sixteen years of age, and adult metachromatic leukodystrophy. associated with onset of manifestation at the age of over sixteen years.

Children with metachromatic leukodystrophy typically experience declines in motor function, and children with late infantile metachromatic leukodystrophy have a five-year survival rate of only approximately 52% (Mahmood A. et al. J. Child Neurol. 2010;25:572-80 ). There are currently no approved pharmacological treatments.

This example describes a non-randomized, open-label, phase 1/2 dose-escalation clinical trial to evaluate the safety and efficacy of recombinant human arylsulfatase A (ASA) in children with metachromatic leukodystrophy (MLD), such as those with late infantile or juvenile metachromatic leukodystrophy. To evaluate efficacy, the effect of recombinant human ASA treatment on gross motor function, brain injury involvement, and CSF sulfatide levels was analyzed.

Eighteen male or female patients were included in the study and met all of the following selection criteria: diagnosis of MLD confirmed based on ASA deficiency (using a white blood cell test) and elevated levels of sulfatides in the patient's urine; the appearance of the first symptoms of the disease at the age of 30 months or earlier; walking at the time of selection (eg, with a minimum required level of function, defined as the ability to stand and walk 10 steps forward while holding one hand); age less than twelve years at

- 32 043909 moment of switching on; and the presence of neurological signs of MLD on screening examination.

Exclusion criteria included: a history of hematopoietic stem cell transplantation; the presence of any known or suspected hypersensitivity to anesthesia or an unacceptably high risk of complications during anesthesia due to a compromised airway or other conditions; the presence of any other medical condition, serious intercurrent illness or debilitating circumstance that would prevent participation in the study. The study design is shown in Fig. 1.

After inclusion, patients were screened to assess their eligibility based on the criteria outlined above and to obtain informed consent. Pre-surgical safety assessments regarding surgical implantation of IDDD can also be performed. Standard institutional surgical procedures were followed.

Patients were enrolled in one of three escalating dose cohorts (n=6 patients per 10, 30, or 100 mg dose cohort) of recombinant human ASA administered intrathecally. If necessary, patients received sedation at the discretion of the administerer. The basis for any dose escalation in newly enrolled patients to the next level was review of the safety data obtained for patients in the lower dose cohort by an independent safety data monitoring committee and representatives of Shire Human Genetic Therapies, and only after patients had received at least two doses. In table Figure 5 shows the demographic characteristics of the patients participating in the study.

Table 5

Characteristic 10 mg rhASA (n=6) 3 0 mg rhASA (n=6) 100 mg rhASA (n=6) Age at inclusion, months Mean ± standard deviation 31.5 ± 11.5 47.3 ± 20.2 52.3 ± 31.1 Range 23-54 31-81 23-107 Boys, η 3 3 5 Ethnicity, η White 5 4 2 Asians 0 0 4 Other 1 2 0

Briefly, the port was placed subcutaneously over the rib, with the catheter placed in the spinal canal at the lumbar level. If the device stopped functioning at any point in time, it was removed and replaced. PORT-A-CATH II® or SOPH-A-PORT® Mini S was used, the latter of which was better tolerated. (See Table 6.) A series of CSF assessments were completed at baseline and at each predose study visit. Dosing of recombinant human ASA was administered via implanted IDDD once every two weeks for 38 weeks (starting at week 0) for a total of 20 injections. IDDD was used for both CSF sampling and recombinant human ASA injections. Samples for safety/efficacy measurements were collected biweekly before dosing.

Safety.

In table Table 6 lists the adverse events reported in the study, with the numbers shown representing the number of patients. As shown in table. 6, There were no treatment-related serious adverse events, deaths, or premature study terminations in any of the three cohorts (10, 30, or 100 mg dose).

Table 6

Reported phenomenon 10 mg rhASA (n=6) 3 0 mg rhASA (n=6) 100 mg rhASA (n=6)

- 33 043909

Any adverse event (AE) 6 6 6 Any AE related to IDDD 3 3 4 Death 0 0 0 Premature termination of the study due to AE 0 0 0 Any serious ^AE 5 4 4 Any AE related to treatment ³ 3 4 4 Blood and lymphatic system disorders 0 2 0 General disorders and reactions at the injection site 3 1 0 Laboratory and instrumental data (results of clinical and laboratory studies) ¹³ 3 1 1 Skin and subcutaneous tissue disorders 2 2 0

^a Events according to organ system class are listed if they are observed in at least two patients in any group.

^b Includes elevated alanine aminotransferase, blood pressure, eosinophil count, and γ-glutamyltransferase levels. AE - adverse events; IDDD - intrathecal drug delivery device; rhASA - recombinant human arylsulfatase A.

Table 7

Serious AE associated with IDDD PORT-A-CATH II® SOPHA-A-PORT® Mini S 10 mg rhASA (n ⁼ 6) 30 mg rhASA (n=5) ^a 100 mg rhASA (n=6) Device failure 2 0 1 Device offset 1 1 0 Device malfunction 1 0 0 Device clogging 1 0 0 Effusion at the implantation site 1 0 1 Infection at the implantation site 0 0 1 Number of patients >1 serious AE associated with IDDD reported by four patients All serious IDDD-related AEs reported one patient

^a One patient was implanted with SOPH-A-PORT® Mini S at baseline. AE - adverse events; IDDD - intrathecal drug delivery device; rhASA - recombinant human arylsulfatase A.

Motor function.

Motor function was assessed using the Gross Motor Functioning Test 88 (GMFM-88), a clinical instrument designed to quantify changes in gross motor function in children. Items in the GMFM-88 include activities involving lying, rolling, walking, running, and jumping skills.

Changes in GMFM-88 total score across all treatment groups at week 40 are shown in FIG. 2. At week 40, the deterioration in motor function was quantitatively lower in the 100 mg group than in the other groups (lower doses), although there were no statistically significant differences between groups.

However, there was a statistically significant relationship between baseline GMFM-88 total score and treatment effect (assessed by change in GMFM-88 score from baseline) (p=0.0067 in ANCOVA; see Figure 3). At 40 weeks, among children with baseline GMFM-88 total scores greater than 40%, less deterioration in motor function was observed in those children treated with 100 mg rhASA than in the other groups. (Cm.

-34043909 fig. 4.)

Involvement in brain damage.

The metachromatic leukodystrophy severity score, determined by magnetic resonance imaging (MLD MRI), is a quantitative indicator of brain damage involvement. (See, e.g., Eichler et al. AJNR Am. J. Neuroradiol. 2009 Nov; 30(10):1893–7.) MRI data from patients at week 40 was assessed by a single investigator in a blinded manner.

As shown in FIG. 5, at week 40, the MRI severity score of MLD was stable in the 100 mg dose cohort, which showed a slight decrease in score. The change in MRI MLD severity scores in the 100 mg dose cohort was significantly different from the changes observed in the 10 and 30 mg dose cohorts, which were characterized by an increase in MRI MLD severity scores over the course of treatment.

Cerebrospinal fluid sulfatide levels.

Concentrations of sulfatide, a metabolite that accumulates in patients with metachromatic leukodystrophy, were studied in the cerebrospinal fluid of patients. As shown in FIG. 6, small decreases in sulfatide concentrations were observed in all cohorts at 40 weeks.

Discussion.

From the results described in this example, it is clear that intrathecal administration of recombinant human ASA to patients with MLD has an acceptable safety profile and stabilizes the disease and/or slows its progression. Since there are currently no available treatments for this progressive and ultimately fatal disease, intrathecal administration of recombinant human ASA represents a promising approach for the treatment of MLD.

Example 2: Additional evaluation of the safety and efficacy of recombinant arylsulfatase A for the treatment of metachromatic leukodystrophy.

Patients with late infantile or juvenile metachromatic leukodystrophy were enrolled in a clinical trial whose design and inclusion and exclusion criteria were similar to those described in Example 1. To further evaluate the safety of recombinant human ASA at specific doses, patients were physically and neurologically examined and monitored indicators of vital functions. In addition, a battery of tests was performed, including urinalysis, serum chemistry panel, serum sulfatide levels, complete blood count, routine cerebrospinal fluid tests and cerebrospinal fluid biomarker tests, and electrocardiography. In addition, concentrations of anti-rhASA antibody and/or levels of metabolites (eg, sulfatide, liosulfatide, and/or N-acetylaspartate) in various fluids, such as cerebrospinal fluid, urine, and blood, were measured. Metabolite levels can also be assessed in the deep white matter of the brain using proton magnetic resonance spectroscopy.

To assess efficacy, one or more changes in GMFM-88 score from baseline, motor nerve conduction measures (such as nerve conduction velocity, total motor nerve action potential, and distal latency), sensory nerve conduction measures (such as nerve conduction velocity, amplitude and distal latency), magnetic resonance imaging and/or magnetic resonance spectroscopy findings to identify MLD-related abnormalities, somatosensory-evoked potential values, functional endoscopic assessment of swallowing, and auditory evoked potential in the brainstem.

Example 3: Decrease in Cerebrospinal Fluid Sulfatide Levels over Time.

The clinical study enrolled children and administered 10, 30, or 100 mg of recombinant human arylsulfatase A by intrathecal administration once every two weeks for 40 weeks. In fig. Figure 7 presents data from several children per cohort and includes data from two pairs of siblings (one pair on the 30 mg dose and the other pair on the 100 mg dose). As shown in FIG. 7, there was a general trend toward decreasing cerebrospinal fluid sulfatide levels over time, particularly noticeable in children given the 30 and 100 mg doses. Moreover, in children administered 100 mg of recombinant human arylsulfatase A, sulfatide levels eventually decreased such that they did not exceed the upper limit of normal ranges.

Example 4 Intrathecally delivered rhASA was well tolerated for 104 weeks.

The three cohorts described in Examples 1-3 were eligible for a long-term extension study that included safety analyzes and other outcome measures up to week 104 (including the initial 40-week dose escalation study (Fig. 8)). Children with MLD were eligible for inclusion in the initial 40-week study if they were less than 12 years of age; They had their first symptoms of MLD at 30 months of age or earlier and were able to walk 10 steps while holding one arm at the time of screening. Eligible patients were sequentially enrolled in one of three escalating dose cohorts (cohort 1, 10 mg rhASA; cohort 2, 30 mg rhASA; cohort 3, 100 mg rhASA). Each patient was surgically implanted with an intrathecal drug delivery device (IDDD) before receiving their first dose. Eleven patients were implanted with the PORT-A-CATH II (Smiths Medical ASD, Inc., St. Paul, MN, USA) during the initial phase of the study, and eight patients were implanted with the SOPH-A-PORT Minis (Sophysa, Opce,

France) (including one patient who switched to this device after initial PORT-A-CATH II implantation).

Children received intrathecal injections of rhASA once every 2 weeks. After the initial 40-week study, the dose of rhASA was individually increased in cohorts 1 and 2 (cohort 1, stepwise), with all patients ultimately receiving 100 mg of rhASA in the current study. The decision to switch to a higher dose was based on review of safety data by the data monitoring committee after patients had received at least two doses of the previous dose level. The primary outcome measure was the safety of rhASA and IDDD. Other primary outcomes assessed at week 104 included change in motor function as assessed by the Gross Motor Functioning Measure-88 (GMFM-88) total score (range 0100%); change in MLD severity assessed by magnetic resonance imaging (MRI) and similar scoring method by Eichler et al. (range 0-34, with higher scores indicating greater disease severity) (see, for example, Eichler et al. AJNR Am J Neuroradiol. 2009 Nov; 30(10):1893-7); changes in N-acetylaspartate (NAA) levels in brain white matter assessed by magnetic resonance spectroscopy and changes in cerebrospinal fluid (CSF) sulfatide concentrations. In table Figure 8 shows the demographic characteristics of the patients participating in the study.

__________Table 8. Patient Demographic Characteristics__________

Characteristic Cohort 1 Cohort 2 Cohort 3 (n=6) (n=6) (n=6) Age at inclusion, months 31.5 ± 11.5 47.3 ± 20.2 52.3 ± 31.1 (26, 5, 23-54) (39, 5, 31-81) (41, 0, 23-107) Boys, η 3 3 5 Ethnicity, p ^a White Asian Other 5 4 2 0 0 4 12 0 Age at onset of MLD symptoms, months 17.3 ± 7.1 23.3 ± 3.3 24.0 ± 4.5 (17.0, 6-27) (24.0, 18-28) (24.5, 16-30) Duration of treatment, ^weeks 137.2 ± 63.5 134.7 ± 12.2 111.2 ± 5.2 (159, 9, 28.3s- (128.9, (110, 6, 106.1- 195, 9) 122.1-150, 1) 117, 1)

Data for age at enrollment, age at symptom onset, and duration of exposure are shown as mean ± standard deviation (median, range).

aChildren were included in the study in five countries: France (n=7), Denmark (n=4), Australia (n=3), Germany (n=2) and Brazil (n=2). Two patients from Australia moved to Japan during the extension study.

^b Time from the first dose administered to the last administered dose of rhASA.

^c One child left the 40-week dose escalation study prematurely due to lack of efficacy. MLD, metachromatic leukodystrophy; rhASA, recombinant human arylsulfatase A.

Safety.

A summary of adverse events (AEs) noted up to week 104 is presented in Table. 9. Eleven patients experienced at least one IDDD-related AE, and 14 patients experienced at least one serious AE (SAE). Six patients experienced at least one IDDD-related SAE. Thirteen patients experienced at least one AE that the investigators considered to be related to study treatment, most commonly fever (n=6).

- 36 043909

Table 9. Summary of adverse events observed ______________ up to week 104_____________

Reported phenomenon Cohort 1 (n=6) Cohort 2 (n=6) Cohort 3 (n=6) ANY AE 6 6 6 Any AE related to IDDD 5 2 4 Death 0 1 0 Premature termination studies due to AE 0 0 0 Any SAE ^a 5 5 4 Spasm 2 2 1 Dehydration 1 1 1 Dysphagia 2 1 1 Fibrillary spasm 2 1 3 Muscle spasticity 2 2 0 Nasopharyngitis 2 1 0 Fever 2 0 1 Epileptic state 0 1 2 Viral infection 1 1 1 Any SAE associated with IDDD ^b 4 1 1 Device offset 0 1 0 Device failure 2 1 1 Functional failure devices 2 0 0 Eruption at the implantation site 1 0 1 Infection at the implantation site 0 0 1 Any AE related to treatment ⁰ 4 5 4 Blood and lymphatic system disorders 0 2 0 Eosinophilia 0 2 0 Leukocytosis 0 1 0 General disorders and reactions at the injection site 4 2 0 Fever 4 2 0 Discomfort Laboratory and 0 1 0 instrumental data (results of clinical laboratory research) 4 1 1 Elevated γglutamyltransferase levels 2 0 1 Elevated alanine aminotransferase levels 1 0 0 High blood pressure 1 0 0 Increased body temperature 1 0 0 Increased number of eosinophils 0 1 0 Skin and subcutaneous tissue disorders 2 2 0 Rashes 2 1 0 Hirsutism 0 1 0

Data are shown as number of patients.

^a SAEs according to preferred terms are reported if they were observed in at least three patients in the study. All SAEs were considered unrelated to treatment;

^ъ Cohort 1 and five patients from Cohort 2 received PORT-A-CATH II at baseline. All patients in cohort 3 and one patient in cohort 2 received SOPH-A-PORT Mini S;

^c AEs according to system organ class are reported if they were observed in at least two patients in any group. Preferred terms are shown for each organ system class. AE, adverse event; IDDD, intrathecal drug delivery device; SAE, serious adverse event

Motor function.

The mean GMFM-88 total score, which decreased across all cohorts from baseline to week 104, is shown in Fig. 9A. Two infants in Cohort 3 (aged 23 and 107 months at the start of treatment) showed evidence of stabilization in their motor function, with absolute GMFM-88 total scores greater than 40% up to 104 weeks shown as stars in Fig. . 9B. At 104 weeks, one of these infants (23 months old at the start of treatment) had a GMFM-88 total score of 94.3%. This patient's older sibling (also from Cohort 3), who was treated at 42 months, had a GMFM-88 total score of 12.2% at approximately the same age, and at 104 weeks the score was 13.7%. Based on scores from 34

- 37 043909 healthy children aged 0-6 years, the mean GMFM-88 total score in healthy children <30 months was determined to be >90% (see, for example, Sessa M et al. Lancet 2016;388:476-87) . The mean ± standard deviation change in GMFM-88 total score was calculated from baseline to week 104. After week 40, patients in cohorts 1 and 2 were individually increased in their doses (cohort 1, stepwise), with all patients ultimately receiving 100 mg rhASA.

Measurements of MLD severity.

The mean MRI severity summary score for MLD increased from baseline to week 104 in cohorts 1 and 2; however, it appeared to be sustained over the same period in Cohort 3 (Figure 10). Mean ± standard deviation of MRI MLD severity summary score at baseline (score ranges 0 to 34, with higher scores indicating greater disease severity). The effect of intrathecally delivered rhASA on the mean NAA/creatine metabolite ratio in brain white matter was assessed over 104 weeks of treatment. The mean white matter NAA/creatine ratio of the frontal lobe (Figure 11A) and frontal and parietal lobes (Figure 11B) was stable over 104 weeks in Cohorts 2 and 3, but trended toward a decrease in Cohort 1. A decrease in the NAA/creatine ratio indicates neuronal loss and progression of MLD. In four healthy children aged 2.5-6 years, the NAA/creatine ratio in periventricular white matter was 1.51-2.30 (see, for example, Assadi M et al. J Cent Nerv Syst Dis 2013;5:25-30 ).

CSF sulfatide concentrations.

Mean CSF sulfatide concentrations decreased from baseline to week 104 in all cohorts, but were within the normal range in cohort 3 (<0.11 μg/mL based on CSF samples from 60 pediatric patients) from week 15 (Fig. . 12). Baseline CSF sulfatide levels were lower in cohort 3 than in the other two cohorts.

Brief description of the additional study.

Intrathecally delivered rhASA was well tolerated in children with MLD. Although there was an overall decline in motor function over the 104 weeks of treatment, there was evidence of response to treatment in patients receiving 100 mg rhASA from the start of the study (cohort 3). In Cohort 3, there was minimal change in mean MRI severity score of MLD and white matter NAA levels. Stabilization of motor function was observed in two patients from cohort 3. Response to treatment was not observed in cohorts 1 and 2. One child in the study may have a form not typical of late infantile MLD but more typical of juvenile MLD. These results provide the basis for the ongoing development of intrathecally delivered rhASA as a potential treatment for patients with late infantile MLD. An additional six children (cohort 4) were subsequently enrolled in the study and are currently receiving 100 mg rhASA biweekly.

The singular forms used herein in the description and claims, unless expressly stated to the contrary, are to be understood to include references to the plural. Claims or specifications that include or between one or more members of a group are considered to satisfy the requirements if one, more than one, or all members of the group are present, used, or otherwise relate to the specified product or process, unless otherwise stated or otherwise obvious from the context. The present invention includes embodiments in which exactly one member of the group is present, used or otherwise related to the specified product or method. The present invention also includes embodiments in which more than one or all members of the group are present, used or otherwise related to the specified product or method. It is further understood that the present invention covers all variations, combinations and combinations in which one or more limitations, elements, conditions, descriptive terms, etc. of one or more of the listed claims are inserted into another claim dependent on the same independent claim (or any other claim, as applicable), unless otherwise indicated or unless one of ordinary skill in the art it will not be obvious in this area that a contradiction or inconsistency will arise. If elements are presented in list form (eg, a Markush group or similar format), it should be understood that each subgroup of elements is also disclosed, and any element(s) may be removed from the group. In general, it should be understood that if the present invention or aspects of the present invention are/are designated as containing/containing specific elements, features, etc., then certain embodiments of the present invention or aspects of the present invention consist or are substantially composed of such elements, features, etc. For purposes of simplicity, such embodiments have not been specifically set forth in such detail herein in every case. It should also be understood that any embodiment or aspect of the present invention may be expressly excluded from the claims regardless of whether a specific exclusion is set forth in the specification. Publications, websites and other references incorporated herein by reference to describe the prior art of the present invention and to provide additional information regarding its practical implementation are incorporated herein by reference.

Sequence Listing <110> Shayer Human Genetic Therapies, Inc.

<120> METHODS AND COMPOSITIONS FOR DELIVERY OF ARYLSULPHATASE A TO THE CNS <130> SHR-1096WO <150> 62/296563 <151> 2016-02-17 <150> 62/453,864 <151> 2017-02-02 <160>2 <170> PatentIn version 3.5 <210>1 <211>489 <212> PROTEIN <213> Homo sapiens <400>1

Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly

5 1015

Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp 20 2530

Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val 35 4045

Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro 50 5560

Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly 65 70 7580

Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg 85 9095

Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro

100 105110

Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe Leu Gly

115 120125

Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr Cys Phe

- 39 043909

Gly Gly Cys

130

135

140

Pro 145

Ile

Pro

Asp

His

Gly 225

Gly

Leu

Arg

Glu

Ala 305

Thr

Gly

Gln

Phe

Pro Ala Thr

Pro Cys Asp

150

Asp Gln Gly 155

Leu Val Pro

160

Pro Leu Leu

Gly Leu Glu

180

Ala Gln Arg

195

Thr His Tyr 210

Arg Gly Pro

Thr Leu Met

Val Ile Phe

260

Gly Gly Cys

275

Gly Gly Val 290

Pro Gly Val

Leu Ala Ala

Phe Asp Leu

340

Ser Leu Phe

355

Ala Val Arg 370

Ala Asn Leu 165

Ala Arg Tyr

Gln Asp Arg

Pro Gln Phe

215

Phe Gly Asp

230

Thr Ala Ile 245

Thr Ala Asp

Ser Gly Leu

Arg Glu Pro

295

Thr His Glu

310

Leu Ala Gly 325

Ser Pro Leu

Phe Tyr Pro

Thr Gly Lys

375

Ser Val Glu

170

Met Ala Phe

185

Pro Phe Phe 200

Ser Gly Gln

Ser Leu Met

Gly Asp Leu

250

Asn Gly Pro

265

Leu Arg Cys 280

Ala Leu Ala

Leu Ala Ser

Ala Pro Leu

330

Leu Leu Gly

345

Ser Tyr Pro 360

Tyr Lys Ala

Ala Gln Pro

Ala His Asp

Leu Tyr Tyr

205

Ser Phe Ala

220

Glu Leu Asp 235

Gly Leu Leu

Glu Thr Met

Gly Lys Gly

285

Phe Trp Pro

300

Ser Leu Asp 315

Pro Asn Val

Thr Gly Lys

Asp Glu Val

365

His Phe Phe

380

Pro Trp Leu

175

Leu Met Ala 190

Ala Ser His

Glu Arg Ser

Ala Ala Val

240

Glu Glu Thr

255

Arg Met Ser 270

Thr Thr Tyr

Gly His Ile

Leu Leu Pro

320

Thr Leu Asp

335

Ser Pro Arg 350

Arg Gly Val

Thr Gln Gly

- 40 043909

Ser Ala His Ser Asp 385

Ser Leu Thr Ala His

405

Pro Gly Glu Asn Tyr

420

Glu Val Leu Gln Ala

435

Thr Thr 390

Glu Pro

Asn Leu

Leu Lys

Ala Asp

ProLeu

Leu Gly

425

Gln Leu 440

Pro Ala

395

Leu Tyr 410

Gly Val

Gln Leu

Cys His

Asp Leu

Ala Gly

Leu Lys

445

Ala Ser

Ser Lys

415

Ala Thr 430

Ala Gln

Asp Ala Ala Val Thr 450

Phe Gly

455

Pro Ser

Gln Val

Ala Arg 460

Gly Glu

Pro Ala Leu Gln Ile 465

Cys Cys 470

His Pro

Gly Cys

475

Thr Pro

Arg Pro

Ser 400

Asp

Pro

Leu

Asp

Ala 480

Cys Cys His Cys Pro

485

Asp Pro

His Ala <210>2 <211>507 <212> PROTEIN <213> Homo sapiens <400>2

Met Gly Ala Pro Arg 15

Ser Leu

Leu Leu

Val Ala Arg Pro Pro

Asn Ile

Val Leu

Tyr Gly Asp Leu Gly 35

Cys Tyr

Gly His 40

Leu Asp Gln Leu Ala 50

Ala Gly 55

Gly Leu

Ala Leu 10

Ile Phe

Pro Ser

Arg Phe

Ala Ala

Ala Asp

Ser Thr

Thr Asp 60

Pro Val Ser Leu Cys 65

Thr Pro 70

Ser Arg

Ala Ala

Leu Leu

Gly Leu 15

Asp Leu 30

Thr Pro

Phe Tyr

Thr Gly

Leu Pro Val Arg Met 85

Gly Met

Tyr Pro

Gly Val 90

Leu Val

Pro Ser

Arg Gly Gly Leu Pro

100

Leu Glu

Glu Val

105

Thr Val

Ala Glu

Val Leu

110

Ala

Gly

Asn

Val

Arg 80

Ser

Ala

- 41 043909

Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys

115 120

Trp His Leu Gly Val

125

Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln

130 135

Gly Phe His Arg Phe 140

Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro

145 150 155

Cys Gln Asn Leu Thr

160

Cys Phe Pro Pro Ala

Thr Pro

165

Cys Asp Gly Gly Cys Asp Gln Gly Leu

170 175

Val Pro Ile Pro Leu Leu Ala Asn Leu

180 185

Ser Val Glu Ala Gln Pro Pro

190

Trp Leu Pro Gly

195

Met Ala Asp Ala

210

Ser His His Thr 225

Leu Glu Ala Arg Tyr Met Ala Phe Ala

200205

Gln Arg Gln Asp Arg Pro Phe Phe Leu

215220

His Tyr Pro Gln Phe Ser Gly Gln Ser

230235

His Asp Leu

Tyr Tyr Ala

Phe Ala Glu

240

Arg Ser

Gly Arg Gly Pro Phe Gly Asp Ser Leu Met

245 250

Glu Leu Asp Ala

255

Ala Val

Gly Thr Leu Met 260

Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu

265 270

Glu

Met

Thr 305

His

Leu

Thr Leu 275 Val Ile Phe Thr Ala 280 Asp Asn Gly Pro Glu 285 Thr Met Arg Ser 290 Arg Gly Gly Cys Ser 295 Gly Leu Leu Arg Cys 300 Gly Lys Gly Thr Tyr Glu Gly Gly Val 310 Arg Glu Pro Ala Leu 315 Ala Phe Trp Pro Gly 320 Ile Ala Pro Gly 325 Val Thr His Glu Leu 330 Ala Ser Ser Leu Asp 335 Leu Pro Thr Leu 340 Ala Ala Leu Ala Gly 345 Ala Pro Leu Pro Asn 350 Val Thr

Leu Asp Gly Phe Asp Leu Ser Pro

355 360

Leu

Leu Leu Gly Thr Gly Lys Ser 365

- 42 043909

Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg

370 375380

Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr

385 390 395400

Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala

405 410415

Ser Ser Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser

420 425430

Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala

435 440445

Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala

450 455460

Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly

465 470 475480

Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg

485 490495

Pro Ala Cys Cys His Cys Pro Asp Pro His Ala

500505

Claims (15)

CLAIM 1. A method of treating metachromatic leukodystrophy syndrome (MLD), comprising the step of intrathecal administration to a subject in need of treatment of a recombinant arylsulfatase A (ASA) enzyme at a therapeutically effective dose of at least 100 mg and with an interval between administrations for a treatment period of at least 2 a year sufficient for: (a) improvement, stabilization, or reduction in the rate of deterioration of one or more motor functions compared to baseline; (b) to reduce the levels of a biomarker accumulating in MLD in a physiological fluid selected from the group consisting of cerebrospinal fluid, urine, blood and serum, compared with the baseline biomarker level; (c) increases in MLD-depleted biomarker levels in brain tissue compared to baseline biomarker levels; or stabilization or reduction of brain damage involvement compared to baseline, as assessed by the MLD severity score determined by MRI (magnetic resonance imaging). 2. The method according to claim 1, where the introduction of the recombinant ASA enzyme further leads to improvement, stabilization or reduction in the rate of deterioration of one or more cognitive, adaptive and/or executive functions. 3. The method of claim 2, wherein the one or more motor functions include a gross motor function, where optionally the gross motor function is assessed using a Gross Motor Functioning Test (GMFM), where optionally the GMFM test is a GMFM-88, where optionally GMFM-88 raw score above or below 40%. 4. Method according to any one of claims 1-3, where the introduction of the recombinant ASA enzyme leads to: (a) a decrease in the GMFM-88 score by less than 10, 20, 30, 40 or 50%; (b) significant stabilization of the GMFM-88 score; and/or (c) improving the GMFM-88 score. - 43 043909 5. The method of claim 1, wherein the biomarker is selected from the group consisting of sulfatide, lysosulfatide, and combinations thereof, wherein the biomarker is optionally a sulfatide. 6. The method according to claim 1 or 5, where the physiological fluid is cerebrospinal fluid, where optionally: (a) the baseline cerebrospinal fluid sulfatide level is greater than approximately 0.1 μg/mL, greater than approximately 0.2 μg/mL, or greater than approximately 0.3 μg/mL; and/or (b) administration of the recombinant ASA enzyme results in a decrease in cerebrospinal fluid sulfatide levels by more than about 0.1 μg/ml or by more than about 0.2 μg/ml. 7. Method according to claim 1, where: (a) brain tissue is the deep white matter of the brain; and/or (b) the biomarker is a metabolite, where optionally the metabolite is N-acetyl aspartate, where optionally N-acetyl aspartate levels are assessed using proton magnetic resonance spectroscopy. 8. The method according to claim 1, where the introduction of the recombinant ASA enzyme leads to: (a) a decrease in the subject's MLD severity score, as determined by MRI, compared to baseline; or (b) stabilization of the subject's MLD severity score, as determined by MRI, compared to baseline. 9. The method according to any of the previous claims, wherein the therapeutically effective dose is between 100 and 200 mg. 10. The method according to any of the previous paragraphs, where the interval between administrations is one week, two weeks or one month. 11. Method according to any of the previous paragraphs, where: (a) the subject is a mammal, where the subject is not necessarily a human; and/or (b) the subject's age is sixteen years or less, twelve years or less, nine years or less, six years or less, four years or less, three years or less, two years or less, 18 months or less, 12 months or less, 6 months or less. 12. Method according to any of the previous paragraphs, where: (a) the subject (i) exhibited at least one symptom of metachromatic leukodystrophy or (ii) did not exhibit any symptoms of metachromatic leukodystrophy; and/or (b) the subject has (i) been diagnosed with metachromatic leukodystrophy or (ii) has been identified as being at risk for developing metachromatic leukodystrophy. 13. Method according to any of the previous paragraphs, where: (a) arylsulfatase A is administered into the spinal canal, where optionally arylsulfatase A is administered into the lumbar region, where optionally arylsulfatase A is administered by lumbar puncture; and/or (b) intrathecal administration is accomplished through intermittent or continuous access to an implanted intrathecal drug delivery device (IDDD). 14. Method according to any of the previous paragraphs, where: (a) the treatment period is at least 12 months, at least 24 months; and/or (b) the subject does not experience serious adverse effects associated with administration of recombinant arylsulfatase A. 15. The method according to any of the previous claims, wherein the recombinant ASA enzyme contains the amino acid sequence: (a) at least 85% identical at the amino acid level to SEQ ID NO: 1; (b) at least 90% identical at the amino acid level to SEQ ID NO: 1; (c) at least 95% identical at the amino acid level to SEQ ID NO: 1; (d) at least 98% identical at the amino acid level to SEQ ID NO: 1; (e) being SEQ ID NO: 1; (f) containing no more than four mismatches with SEQ ID NO: 1; (g) containing no more than three discrepancies with SEQ ID NO: 1; (h) containing no more than two mismatches with SEQ ID NO: 1; and/or (i) containing no more than one mismatch with SEQ ID NO: 1.