Delivery of therapeutic compounds to the brain and other tissues

[0001] The invention relates to intrathecal (IT) administration of a recombinant enzyme in the treatment of lysosomal storage disorders. In addition, induction of specific antigen tolerance is considered prior to intrathecal administration of the replacement enzyme.

BACKGROUND OF THE INVENTION [0002] The brain is shielded from potentially harmful substances by the blood-brain barrier (BBB). The septum between blood and brain microcirculation consists of a capillary layer of endothelium surrounded by a basal membrane and closely related helper cells (pericytes, astrocytes). The capillary endothelium of the brain is less permeable to low molecular weight solutes compared to other endothelial capillaries due to the apical syndrome of tight junction between membranes of adjacent cells, referred to below as closing rims. In addition to reduced passive diffusion, brain endothelial capillaries also show less liquid phase pinocytosis than other endothelial cells. The brain's capillaries have several windows and several endocytic vesicles, compared to the capillaries of other organs (see Pardridge, J. Neurovirol. 5: 556-569 (1999)). There is little BBB transport of large hydrophilic molecules, in addition to certain specific proteins, such as transferrin, lactoferrin, and low density lipoproteins that are taken up by receptor-mediated endocytosis (see Pardridge, J. Neurovirol. 5: 556-569 (1999)) ; Tsuji and Tamai, Adv. Drug Deliv. Rev. 36: 277-290 (1999); Kusuhara and Sugiyama, Drug Discov. Today 6: 150-156 (2001); Dehouck, et al. J. Cell. Biol. 138: 877-889 (1997); Fillebeen, et al. J. Biol. Chem. 274: 7011-7017 (1999)).

[0003] The blood brain barrier (BBB) ​​also impedes the access of beneficial active agents (e.g. therapeutic agents and diagnostic agents) to central nervous system (CNS) tissues, which requires the use of carriers for their transport. The permeability of the blood-brain barrier is often a limiting factor in the rate of drug or peptide penetration in the CNS (see Pardridge, J. Neurovirol. 5: 556-569 (1999); Bickel, et al., Adv. Drug Deliv. Rev. 46: 247- 279 (2001)). For example, the management of neurological symptoms of lysosomal storage diseases (LSD) is significantly hampered by the inability of therapeutic enzymes to gain access to brain cell lysosomes. LSDs are characterized by the lack or reduced activity of specific enzymes within cell lysosomes, which results in the accumulation of non-degraded "deposited material" in the intracellular lysosome, edema and lysosome damage and ultimately cell and tissue damage. Intravenous enzyme replacement therapy (ERT) is beneficial for LSD (e.g. MPS I, MPS II). However, BBB blocks the slow transfer of many agents from the blood to the brain and it is not expected that LSD, which have significant neurological sequelae (e.g. MPSI, MPS III, MLD, GM1) will respond to intravenous

ERT. With such diseases, a method for BBB delivery of the replacement enzyme and in the lysosomes of affected cells would be highly desirable.

[0004] There are several ways to circumvent BBB to increase delivery of the active agent to the brain, including direct intracranial injection, transient BBB permeabilization, and modification of the active agent to alter tissue distribution. Direct injection of the active agent into the brain tissue completely bypasses the vessels, but is primarily associated with the risk of complications (infection, tissue damage) occurring with intracranial injections and poor diffusion of the active agent from the site of administration. BBB permeabilization entails nonspecific BBB exposure associated with intravenous active agent injection and is accomplished by loosening the closing rims as a result of hyperosmotic shock (e.g. intravenous mannitol). High plasma osmolarity leads to dehydration of the capillary endothelium with partial breakdown of the closing rims, low selectivity of the types of blood derivatives that gain access to the brain under these conditions, and damage during the lifetime existing treatment regimen.

[0005] The distribution of the active agent in the brain can also be increased by transcytosis, the active transport of certain proteins from the luminal space (blood side) to the abluminal area (brain side) of BBB. Transcytosis pathways differ from other types of follicular movement in the capillary endothelial cell and transport can occur without altering the materials transferred. Trancipitosis is a cell-specific process mediated by receptors on the BBB endothelial surface. Attachment of the active agent to transcytosis protein (vector or vehicle) is expected to increase distribution of the active substance to the brain. In transcytosis it is assumed that the vector has a dominant effect on the distribution of the combined pair. Vector proteins include receptors directed to receptors on cerebral vascular endothelial cells (Pardridge, J. Neurovirol. 5: 556-569 (1999)) and ligands to these receptors (Fukuta et al., 1994, Pharm Res. 1994; 11 (12): 1681-8; Broadwell, et al., Exp Neurol. 1996; 142 (1 ): 47-65)). Antibody vectors are transported through the capillary endothelium in the process of adsorptive endocytosis (non-specific endocytosis of the membrane phase) and are much less efficiently transported than the actual receptor ligands that exceed BBB via an energy-dependent saturated mechanism (Broadwell, et al. Exp Neurol. 1996; 142 (1): 47-65).

[0006] Direct administration of proteins to brain substances does not achieve a significant therapeutic effect due to diffusion barriers and the limited volume of therapeutic that can be administered. Convection diffusion was analyzed by catheters placed in the brain parenchyma using slow long-term infusions (Bobo, et al., Proc. Natl.Acad.Sci. USA 91, 2076-2080 (1994); Nguyen, et al. J. Neurosurg . 98, 584-590 (2003)), but no approved therapy currently uses this approach for long-term treatment. In addition, placement of intracerebral catheters is very invasive and less desirable as a clinical alternative.

[0007] Intrathecal (IT) injection or administration of proteins into the cerebrospinal fluid (PMR) has also been used, but they have only resulted in modest success in several cases of PMR delivery [Dittrich et al., Exp.Neurol. 141: 225-239 (1996); Ochs et al., Amyotroph.Lateral.Scler.Other Motor Neuron Disord. 1: 201-206 (2000); Bowes et al., Brain Res. 883: 178-183 (2000)]. In the case of nerve growth factor (NGF), intracerebroventricular administration did indeed have some beneficial effect on the brain (Koliatsos et al., Exp.Neurol. 112, 161-173 (1991), but showed no significant diffusion into the brain substance. The main challenge in this treatment was the tendency to very tightly bind the lining factor of the chamber, which prevented subsequent diffusion. Currently, there are no approved products for the treatment of cerebral genetic disease by therapeutic administration directly into CSF.

[0008] Challenges in the treatment of the brain with these and other therapeutic agents studied in the past suggest that the diffusion barrier on the brain surface as well as the lack of diffusion and the effectiveness of brain treatment are too much an obstacle to achieving the desired therapeutic effect in the brain for each disease. Preliminary data suggests that intraventricular or intrathecal enzyme treatment is not working enough to be effective, and indeed, no studies of this approach have been published recently and there are no successful cases of this treatment. Intrathecal injection has an advantage over other standard treatment regimens, however, including PMR provides better access to the brain and meninges. PMR covers the brain and provides a large surface area of ​​contact with neurons of the cortex up to 6 mm below the surface, which allows more effective penetration of the therapeutic in the brain tissue.

[0009] Shull et al. (Proc. Natl. Acad. Sci. USA, vol. 94, pp. 12937-12941) refers to the replacement enzyme in the canine model of Hurler syndrome. von Specht et al. (Neurology 29: 848-854, June 1979) relates to a surrogate enzyme in Tay-Sachs disease.

[0010] Liposomal storage disorders affecting the nervous system pose unique challenges in the treatment of these diseases by traditional therapies. Affected individuals often have a large accumulation of glycosaminoglycans (GAGs) in neurons and meninges, leading to mild or severe forms of the disease. Although, brain disease in patients with severe MPS I is characterized by developmental delay, hydrocephalus, severe mental retardation and the final fall and death due to disease symptoms. Benign MPS and brain are characterized by perivascular storage of GAG, hydrocephalus, learning discomfort and compression of the spinal cord due to swelling and scar caused by memory diseases. In patients with MPS I who are affected by meningeal storage, the meninges are blocked, reducing PMR resorption and leading to high pressure hydrocephalus. This abnormal lysosomal storage also leads to thickening and scarring of the meninges due to storage disease.

[0011] Lysosomal storage disorders, Gaucher disease, patients with severe disease (type 2 and type 3) have brain disease and intravenous enzyme therapy is insufficient to effectively and properly treat the brain. Intrathecal and intraparenchymal enzyme therapy with glucocerebrosidase, enzymatic deficiency in Gaucher's disease, has an effect on getting into the brain, but does not effectively cure brain storage (Zirzow et al., Neurochem. Res. 24, 301-305. 1999). No brain disease caused by lysosomal disorder was successfully cured at this time by available means.

[0012] Thus, there is still a need in the art to develop methods for effectively treating lysosomal storage disorders by efficiently administering enzyme replacement therapy. In particular, there is a need for more effective methods of administering compounds and compositions that can more efficiently deliver active agents to the brain and central nervous system for treating lysosomal storage disorders.

SUMMARY OF THE INVENTION [0013] The invention relates to pharmaceutical compositions for use in the treatment of central nervous system symptoms of enzymatic storage diseases. In particular, the invention is based on the discovery that intrathecal delivery of compositions containing enzymes that are deficient or not present at all in lysosomal storage disorders results in a prolonged, long-term clinically useful therapeutic intervention of central nervous system symptoms in such diseases. Thus, the invention is directed to enzymatic replacement therapy for such diseases by intrathecal administration of subjects requiring such treatment into the cerebrospinal fluid.

[0014] Therefore, in one aspect of the invention, there is provided a pharmaceutical composition comprising an enzyme which is (a) deficient in lysosomal storage disease, and (b) comprises or has been designed to contain a residue that enables this enzyme attachment of mannose-6-phosphate receptor (M6P), for use in treating lysosomal storage disease by intrathecal administration to a mammal in an amount effective to alleviate the CNS symptoms of lysosomal storage disease.

[0015] In particularly preferred embodiments, the pharmaceutical compositions of this invention provide intrathecal administration of iduronidase for therapeutic intervention in MPS. This treatment has a beneficial effect on the patient because it reduces or eliminates the storage of glycogen granules in the tissues. In addition, intrathecal injection of the enzyme into the cerebrospinal fluid of both neonatal and adult patients results in therapeutic levels of iduronidase in the brain and reduction or elimination of storage of glycosaminoglycan granules in brain tissue.

[0016] Although some embodiments use iduronidase as a swap / substitution enzyme, it should be understood that the methods of the invention can be used for therapeutic interventions in other diseases that require the administration of another enzyme. For example, the invention also contemplates intrathecal administration of beta-glucuronidase (MPS VII) iduronate sulfatase (MPS II), alpha-N-acetylglucosaminidase (MPS IIIB), arylsulfatase A (MLD), glucocerebrosidase, β-glucosidase or N-acetylgalactosamine 4sulfatase.

[0017] Furthermore, it is believed that the presence on the cell surface of brain cells of the receptor with high affinity for enzyme uptake, even at low enzyme concentrations, will create a concentration gradient in CSF CSF that directs the enzyme to cross the brain surface across the brain-PMR border .

[0018] In preferred embodiments, this invention is directed to a pharmaceutical composition for use in treating a mammal lysosomal storage disease comprising spinal administration to the mammalian central nervous system of a pharmaceutical composition comprising an enzyme that is deficient in lysosomal storage disease in an amount that is effective to relieve symptoms lysosomal storage diseases. Specialists in the field constantly monitor subjects for the symptoms of lysosomal storage disease through routine history assessment, medical examination, echocardiography, electrocardiography, magnetic resonance imaging, polysomnography, skeletal examination, various motion measurements, corneal photographs and skin biopsy (see US Patent Application No. 6,585 971). Any such methods can be used in conjunction with the treatment methods described herein.

[0019] Preferably, the enzyme replacement therapy pharmaceutical composition is administered in an amount effective to reduce the amount of storage granules present in the mammalian brain tissue. More specifically, therapeutic results in reducing GAG build-up in the nerve tissue and / or meningeal tissue of a subject. In certain preferred pharmaceutical compositions of the invention, therapeutic intervention relieves high pressure hydrocephalus associated with lysosomal storage disease. Preferably, intrathecal administration of the enzyme therapy of this invention reduces the edema of the meninges that results from the presence of lysosomal storage granules in the meninges of individuals suffering from lysosomal storage diseases.

[0020] The pharmaceutical compositions of the invention may be used in the treatment of any lysosomal storage disease that manifests in the brain or meninges and requires drug delivery to the brain or meninges. The pharmaceutical compositions of this application achieve a therapeutic effect by crossing / crossing the brain-PMR border and alleviating the harmful effects of lysosomal storage diseases in brain tissue. For example, such disease may include, but not be limited to, aspartylglucosaminuria, cholesterol ester storage disease, Wolman's disease, cystinosis, metachromatic leukodystrophy, Danon's disease, Fabry's disease, Farber lipogranulomatosis, Farber's disease, fucosidosis, galactosialidosis type I / II, diseases Gaucher types I / II / III, Gaucher disease, globoid cell leukodystrophy, Krabbe disease, glycogen storage disease II, Pompe disease, GM1-gangliosidosis types I / II / III, GM2-gangliosidosis type I, Tay Sachs disease, GM2gangliosidosis type II, Sandhoff disease, GM2-gangliosidosis, α -mannosidosis types I / II, β-mannosidosis, metachromatic leukodystrophy, mucolipidosis type sialidosis types I / II mucolipidosis types II / III I-cell disease, mucolipidosis type IIIC pseudo-Hurler polydystrophy, mucopolysaccharidosis type I mucopolysaccharidosis type II, Hunter's syndrome, type IIIA mucopolysaccharidosis, Sanfilippo syndrome, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, Morquio syndrome mucopolysaccharidosis type IVA, Morquio syndrome type Mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly syndrome, type N mucopolysaccharidosis, B-type sulfonidase, lipid deficiency , Niemann-Pick type A / B diseases, Niemann-Pick diseases, Niemann-Pick type C1 diseases, Niemann-Pick type C2 diseases, pycodyzostoses, Schindler's disease types I / II, Schindler's disease and sialic acid storage disease.

[0021] In particularly preferred embodiments, the disease is mucopolysaccharidosis and more preferably the disease is mucopolysaccharidosis I. In some embodiments, the subject with lysosomal storage disease has a reduced normal α-L-iduronidase activity. Activity may be reduced because the enzyme is mutated or absent in the subject. In a particular embodiment, the mammal has about 50% or less normal α-L-iduronidase activity. In other embodiments, the patient has 75% or less normal α-L-iduronidase activity. To treat this deficiency, the methods of the invention may use a pharmaceutical composition that contains a dose of at least about 125,000 units or 0.5 mg / kg of human α-L-iduronidase. Other preferred doses comprise from about 0.01 mg / 15-20 kg patient weight to about 10 mg / 15-20 kg patient weight. The dose may be administered at any convenient dose and in preferentially spaced intervals designated by the treating physician. In some embodiments, enzyme replacement therapy is administered to a patient suffering from lysosomal storage enzyme deficiency.

[0022] In some exemplary embodiments, a pharmaceutical composition comprising a dose of at least about a dose of about 0.01 mg / 15 ml PMR to about 5.0 mg / 15 ml PMR in a human mammal, α-L-iduronidase is administered once week for a patient suffering from its deficiency. Preferably, a pharmaceutical composition containing a dose of about 1 mg / 15 ml PMR in a human mammal α-L-iduronidase is administered once a week to a patient suffering from its deficiency. An exemplary pharmaceutical composition is formulated in a buffer containing 0.58 mg / ml iduronidase in a buffer containing 100 mM sodium phosphate, 150 mM NaCl and 0.001% polysorbate 80.

[0023] The pharmaceutical compositions of this invention may also contain other ingredients such as, for example, human albumin. In particular embodiments, the compositions contain human albumin at a concentration of at least about 1 mg / ml. The compositions may be in the form of buffer solutions, such as, for example, a buffer solution containing a sodium phosphate buffer at a concentration of about 10-50 mM.

[0024] In specific embodiments, the lysosomal storage disorder is MPS 1 and the enzyme is a recombinant iduronidase administered intrathecally in an amount from about 0.5 mg to about 20 mg per kilogram body weight. In particular embodiments, the amount is from about 0.5 mg to about 0.5 mg per kilogram body weight. In particular, it is believed that recombinant iduronidase is administered at a dose of about 1.0 mg to 100 mg, 2.0 mg to 50 mg or 10 mg to 100 mg per kilogram body weight. These are merely examples of iduronidase amounts, and those skilled in the art will understand that these doses may vary depending on the patient's age, patient size, stage of the disease, and the like. In preferred embodiments, the recombinant iduronidase is administered at a dose of about 1.0 mg to 15 mg; 2.0 mg to 10 mg or 10 mg to 5 mg.

[0025] The enzyme in replacement therapy may be produced by any source commonly used for the production of such enzymes. In some embodiments, the enzyme is iduronidase, which is secreted and purified from mammalian cells in culture transfected with a DNA sequence encoding human iduronidase.

[0026] The enzyme delivered by the intrathecal methods of treatment may be administered by any convenient pathway commonly used for intrathecal administration. For example, intrathecal administration may be by slow infusion of at least 0.5 mg / kg of formulation within about an hour. However, it should be understood that the dose may vary from about 0.01 mg / 15-20 kg patient weight to about 10 mg / 15-20 kg patient weight at a similar infusion rate. Preferably, intrathecal administration of enzyme replacement therapy results in the normalization of lysosomal granulation in neuronal and / or meningeal tissue of subjects as discussed above. In particularly preferred embodiments, it is contemplated that granulation storage is ameliorated from neuronal and glial tissues, resulting in a reduction of developmental developmental delay and regression seen in individuals suffering from lysosomal storage disease. Other preferred embodiments lead to the normalization of lysosomal storage granules in meningitis near the arachnoid granules, the presence of which in lysosomal storage disease causes high pressure hydrocephalus. Accordingly, the pharmaceutical compositions are intended for the treatment of such high pressure hydrocephalus associated with lysosomal storage disease. The pharmaceutical compositions of the invention may also be used to treat compression of the spinal cord that results from the presence of lysosomal storage granules near the cervical meninges on the C1-C5 segment or elsewhere in the spinal cord. The pharmaceutical compositions of the invention are also intended for the treatment of cysts that are caused by perivascular lysosomal storage of granules around brain vessels.

[0027] In other embodiments, treatment may also advantageously lead to normalization of liver volume and urinary glycosaminoglycan secretion, reduction of spleen size and occurrence of apnea / hypopnea, increase in growth rate and development in pre-puberty patients, increase in shoulder flexion and extension elbow and knees, and reduction of tricuspid or pulmonary valve regurgitation. Those skilled in the art are particularly recommended in the methods for monitoring such effects described in Example 5, US Patent Application No. 6,585,971, and, more generally, for teaching methods and compositions for the formulation of recombinant iduronidase.

[0028] In preferred embodiments, the therapeutic administration in this application comprises the administration of human recombinant α-L-iduronidase, which reduces lysosomal storage at least in the brain tissue of an individual with lysosomal storage disease. In those preferred aspects of the invention in which iduronidase is administered intrathecally to PMR, the delivered composition contains about 1 mg iduronidase / 20 kg body weight of a mammal that is being treated for MPS. In particular embodiments, the above dose is delivered to 15 ml PMR. At this concentration, it is contemplated that the enzyme concentration will be 18,000 units per ml PMR. It should be understood that the above-mentioned dosage is only an exemplary dosage and it will be understood by those skilled in the art that the dosage may be varied.

[0029] Intrathecal administration may include introducing the pharmaceutical composition into the brain ventricle. Alternatively, intrathecal administration may comprise introducing the pharmaceutical composition into the lumbar region. In yet another alternative embodiment, intrathecal administration includes introducing the pharmaceutical composition into a large reservoir. Any such administration is preferably carried out by bolus injection. Depending on the severity of the patient's symptoms and response to therapy, this bolus injection may be given once a week, once a month, once every two months, once every three months, once every 6 months or once a year. In other embodiments, intrathecal administration is performed using an infusion pump. The pharmaceutical may of course be administered intrathecally for a period of at least several days or alternatively intrathecal administration is continued for a period of at least four weeks. Of course, where administration is by continuous infusion, the rate of administration of the enzyme replacement therapy dose can be significantly reduced compared to the bolus injection.

[0030] In some embodiments, the treatment regimens may be such that intrathecal administration is combined with the systemic administration of a pharmaceutical composition containing this enzyme that is deficient in the disease in combination. In such preferred embodiments, intrathecal administration may be performed one month apart, although other intervals between administrations are also contemplated. Preferably, systemic administration in such combined administration regimen is intravenous administration. In particular embodiments, the pharmaceutical compositions of the invention consider treating lysosomal storage disease by administering an enzyme such as rh-IDU intrathecally to deliver to the CNS and systemically to alleviate the effects of lysosomal storage disease at sites other than the CNS. For example, in specific embodiments, rh-IDU is administered intrathecally at a monthly interval and intravenously in a schedule every two weeks, weekly, daily or every other day. In some embodiments, the subject may be adapted for intrathecal and / or rh-IDU administration by using an immunosuppressive adaptation regimen prior to initiating the therapeutic regimen.

[0031] The pharmaceutical compositions of the invention are beneficial for therapeutic intervention in a human suffering from lysosomal storage diseases.

[0032] In preferred embodiments of the invention, the enzyme is delivered to the cerebrospinal fluid naturally contains or has been designed to contain a component that allows the capture of the enzyme by high affinity. For example, the enzyme contains or has been designed to contain a residue that allows the enzyme to bind to a receptor selected from the mannose-6-phosphate receptor, melanotransferrin receptor, and LRP receptor or another receptor that is commonly expressed on the surface of brain cells. In preferred embodiments, the enzyme contains mannose-6-phosphate residues that allow the enzyme to be captured by a cell that expresses the mannose-6-phosphate receptor. In an alternative embodiment, the enzyme binds naturally or has been designed to have GAG ​​binding capacity. In an alternative embodiment, the enzyme comprises p97, RAP, transferrin or IGF2.

[0033] In some aspects of the invention, patients treated with enzyme replacement therapy for lysosomal storage disease are adapted to tolerate such treatment using tolerance schemes.

[0034] In some embodiments of the invention, the enzyme in enzyme replacement therapy for lysosomal storage disease is such that it naturally contains or is attached to a residue that facilitates high uptake of this enzyme. In preferred embodiments, such an enzyme is iduronidase, or / both recombinant and / or wild type. The residue that facilitates the uptake of the enzyme can be any residue, such as a ligand binding partner or receptor expressed on the cell surface to which treatment is to be targeted. In particularly preferred embodiments, the residue is selected from the group consisting of a mannose-6-phosphate residue, RAP polypeptide, and p97 polypeptide. Other aspects of this invention define methods that further comprise inducing tolerance of a particular antigen prior to enzyme replacement therapy. Such induction of treatment tolerance may utilize administration of an immunosuppressant such as e.g. cyclosporine or alone or in combination with an agent such as azathioprine which may have antiproliferative and / or co-stimulatory signaling blocking effects.

[0035] Particular embodiments contemplate methods for promoting the breakdown of glycosaminoglycans (GAG) in brain cells of a subject having a lysosomal storage disease, a method comprising intrathecally administering to a subject a pharmaceutical composition comprising an enzyme that is deficient in lysosomal storage disease in an amount effective to reduce the amount of GAG present in the cell compared to the amount of GAG present in the cell before administration.

[0036] In specific embodiments in this specification, it should be understood that the methods can be used to reduce GAG ​​storage and / or promote GAG ​​degradation in any brain cell that has abnormal GAG storage. Brain cells can be neurons, glial cells, or lining cells. In specific embodiments, the brain cells may be selected from at least one of a group consisting of neurons, glial cells, microglia cells, astrocytes, oligodermic glial cells, perivascular cells, adventitious cells, meninges, lining cells, arachnoid granulation cells, membranes Arachnoid, dural, meningitis and choroid plexus cells. In preferred embodiments, the brain cell is a meninges cell. It is believed that in some embodiments, the patient has high pressure hydrocephalus and administration reduces the amount of PMR fluid in the subject's meninges. In other embodiments, the subject is anticipated to suffer from spinal cord compression and administration reduces or otherwise ameliorates the symptoms of compression. In preferred embodiments, the therapeutic methods of the invention reduce the number of lysosomal storage granules in a cell, compared to the number of lysosomal storage granules present in a similar cell in the absence of intrathecal administration.

[0037] Another embodiment contemplates a method for reducing meningitis in a subject having a lysosomal storage disease, a method comprising intrathecally administering to a subject a pharmaceutical composition containing an enzyme that is deficient in lysosomal storage disease in an amount effective to reduce meningitis compared to meningitis. the size of the subject's meninges before administration. The subject may be a human.

[0038] Among other preferred aspects of the invention comprising pharmaceutical compositions for use in reducing spinal cord compression in a subject suffering from lysosomal storage disease, the method comprising intrathecally administering to the subject a pharmaceutical composition comprising an enzyme that is deficient in lysosomal storage disease in an amount effective to reduce inflammation meninges, compared to the size of the meninges in the subject prior to administration. In these and other methods of the invention, the subject's motor skills are preferably improved by administering the pharmaceutical composition compared to the motility of the animal prior to administering the pharmaceutical composition.

[0039] The previous paragraphs do not specify all aspects of the invention and additional aspects are described in other sections such as the Detailed Description.

In addition to the above, the invention includes, as an additional aspect, all embodiments of the invention in any way narrower than the changes set out in the individual paragraphs above. At least some aspects of the invention that are described as a genus and it should be understood that each representative of the genus constitutes, individually, an aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS [0041] The following drawings form part of the description and are attached to further illustrate aspects of the invention. The invention may be better understood by reference to the drawing in combination with the detailed description of the individual embodiments presented herein.

Figure 1 shows enzyme levels in the canine brain after spinal injection.

Figure 2 shows levels of rh-iduronidase measured in deep brain and surface brain tissues in dogs.

Figure 3 shows levels of iduronidase activity in the spinal cord and spinal cord tires of MPS I animals treated with iduronidase.

Figure 4 shows a comparison of glycosaminoglycan (GAG) levels in treated animals with MPS I receiving or intrathecal, or IV administration of iduronidase.

Figure 5 depicts electron microscopy (in brain sections) of GAG storage in perivascular macrophage disease in MPS I treated and untreated animals.

Figure 6 is a greater magnification of electron microscopy (brain slices) of GAG storage in perivascular macrophage disease in treated and untreated MPS I animals, which shows that perivascular cells in treated and untreated MPS I animals lack GAG storage.

Figure 7 is a comparison of neuronal disease pathology in treated and untreated MPS I animals, which shows that the treated animals are free of lamellar GAG storage.

Figure 8 is a comparison of brain sections assessed for meningeal disease in treated or untreated MPS I animals, which shows the absence of large GAG ​​filled foam cells in the treated animal's tires compared to controls.

Figure 9 illustrates that brain sections of treated MPS I animals show low lymphocytic infiltration into the meninges.

Figure 10A to 10C includes comparisons of monthly effects vs. weekly spinal rhIDU application.

Figure 10A shows that brain GAG levels were reduced to normal by treatment (* p = 003). Figure 10B shows that GAG levels in the spinal cord were reduced by treatment (p = 0.22). Figure 10C shows that GAG levels in vertebral meninges were reduced by treatment (* p = 0.02).

Figures 11A and 11B show a comparison of GAG between untreated and spinal treated dogs. GAG storage is clearly reduced in perivascular, glial and neocortical cells in treated dogs. Untreated samples (Figure 11A) show foamed, swollen, GAG-loaded cells as contrast with the treated samples shown in Figure 11B, which are thin cells with significantly less storage.

Figure 12A to 12D shows that immune tolerance reduces the inflammatory response to intrathecal administration of rhIDU. Lymphocyte and plasmocyte infiltration develops in treated dogs (Figure 12A and 12C). Pre-conditioning according to the immune tolerance induction scheme greatly reduces this reaction (Figure 12B and 12D).

DETAILED DESCRIPTION OF THE INVENTION [0042] The invention relates to pharmaceutical compositions for use in the treatment of lysosomal storage disorders using spinal injection of enzymes in deficiency in the particular disorder being treated. Pharmaceutical compositions can be coupled with an induction tolerance scheme to provide subjects with more effective treatment.

[0043] The application is based on the finding that spinal delivery of enzyme replacement therapy for lysosomal storage disorders results in an uninterrupted, long-term clinically useful therapeutic intervention of the central nervous system symptoms of such diseases. Enzyme properties such as solubility and attachment to substrates may affect the penetration of the enzyme into brain tissue and the passage of the therapeutic agent across the PMR-brain border.

[0044] Thus, for the first time, the specification details the effective treatment of lysosomal storage diseases within the brain and meninges using recombinant iduronidase, thus showing for the first time that treatment of brain disease and meninges in storage diseases such as MPS I is possible. Assuming that peripheral enzyme therapy for MPS I was approved for human use in 2003, the invention allows immediate transposition for treatment of human patients with MPS I in clinical trials using the same recombinant iduronidase compositions currently approved for peripheral therapy. This represents significant progress in the treatment of lysosomal storage diseases since the brain symptoms of lysosomal storage diseases have been refractory to date. While many of the methods described herein are illustrated using MPS I studies, it is contemplated that the inventions may be extended to treat other lysosomal storage disorders for which enzyme therapies are currently being developed.

[0045] In particular embodiments, the immediate specification specifies for the first time that enzyme iduronidase can penetrate the brain better than other enzymes that have been previously tried. In particularly preferred embodiments, iduronidase is used to treat MPS disorders. Intrathecal administration of iduronidase is contemplated for the ability of this enzyme to bind GAG in brain tissue, which can provide binding sites for the enzyme to be drawn into the tissue fluid space. The presence of mannose-6-phosphate residues on iduronidase allows for high affinity for enzyme uptake from PMR, thus allowing low concentrations of the enzyme in PMR to produce a clinical therapeutic effect. The fact that mannose-6-phosphate residues attach to the high affinity receptor on the surface of almost all cells allows even small amounts of iduronidase to be absorbed by the brain and meninges and being a common feature of lysosomal storage disease in these places.

[0046] Iduronidase has an extremely high affinity for its receptor with half the maximum binding at a concentration of about 1 nanomolar (12 units / ml) and further, assuming half the maximum defect correction at about 1 picomolar, adding even small amounts of enzyme to the PRM space would produce a huge gradient a brain transporting enzyme. In this treatment regimen at a dose of 1 mg in a 20 kg dog with 15 cc PRM, an enzyme concentration in PRM is estimated at about 18,000 units / ml. This concentration is more than 1,000 times the concentration needed to observe uptake and 1,000,000 times the concentration required for half the maximum correction. Thus, even an inefficient process in which only 1% of the enzyme penetrates the brain would result in levels in the brain that are 10 times the uptake constant, a concentration that should provide effective uptake and about 10,000 times above half of the maximum corrective concentration. Assuming this easily achievable gradient, the effects of the enzyme properties at diffusion, and the low concentration needed for uptake and correction, it is shown here that iduronidase effectively treats MPS I symptoms in vivo. Another additional benefit of using spinal iduronidase therapy in the treatment of MPS I is that MPS I disease improves the permeability of the brain surface to enzyme therapy, which makes spinal therapy an attractive way to treat MPS I.

[0047] The importance of the high concentration of the gradient resulting from spinal delivery of the enzyme combined with low but significant enzyme penetration into the brain may have been sufficient to achieve therapeutic efficacy in any lysosomal storage disorder. The effect of this high concentration gradient generated by the binding property of the high uptake receptor iduronidase was not appreciated before the invention and is important for understanding how the enzyme can be forcibly delivered through the lining layer and how many enzymes can now be diffused through the blood barrier brain. Methods and compositions for achieving such correction of iduronidase as well as other enzymes for lysosomal storage diseases are discussed in more detail below.

Definitions [0048] It should also be understood that the terminology used herein is only for the purpose of describing particular embodiments and is not intended to be limited as the field of the invention will be limited only by the appended claims.

[0049] Where a range of values ​​is provided, it is understood that any value intervening up to one-tenth of the lower limit unit, unless the context clearly dictates otherwise, between higher and lower range limits and any other given or intervening value within that given range is covered within the scope of the invention. Higher and lower limits of these smaller ranges may be independently contained in smaller ranges, provided that any specifically excluded limit within the given range.

[0050] Unless otherwise specified, all terms used herein have the meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or corresponding to those described herein can also be used in the practice or testing of the invention, preferred methods and materials are now described.

[0051] It should be noted that the singular "a", "an" and "the" used herein and in the appended claims include plural references, unless the context clearly indicates otherwise.

[0052] By "lysosomal storage disease" is meant any disease resulting from a deficiency of one or more lysosomal enzymes needed to metabolize natural macromolecules. These diseases typically result in the accumulation of non-degraded molecules in lysosomes, resulting in increased numbers of storage granules (also referred to as storage vesicles). These diseases are described in more detail below.

[0053] A "subject" includes any animal that is to be treated using the methods of the invention. Preferably, the subject is a mammal, including, without limitation, humans and non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs and the like. This term does not mean a specific age or gender. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are covered by the term "subject."

[0054] By "therapeutically effective" specification is meant to mean any therapeutic benefit that arises from the treatment methods of the invention. For example, such effects may be beneficial effects that are manifested in the appropriate target tissue, an enzyme that is deficient or lacking in lysosomal disorder in interest, where such beneficial physiological effect is compared to that physiological parameter that is measured in the absence of enzyme replacement therapy. Such therapeutic effect can be any reduction or elimination of one or more clinical or subclinical manifestations of the disease in interest. For example, reducing the number of storage bubbles (also known as storage granules) or eliminating them will provide a therapeutic benefit to the subject being treated. Methods for detecting the presence of storage granules in tissue of interest are well known in the art and described below in the art. Such methods require microscopic examination of tissue sections. See, e.g., Vogler et al. (1990) Am J Pathol 136: 207-217. In addition, reducing the accumulation of substances due to the particular enzyme deficiency in question will also bring therapeutic benefit to the subject being treated. Such substances can be easily detected using known analyzes. For example, MPS VII results in the accumulation of non-degraded glycosaminoglycans (GAGs). GAG levels can easily be measured using methods developed by Farndale et al. (Famdale et al. (1982) Con Tissue Res 9: 247248) and Poorthuis et al. (Poorthuis et al. (1994) Pediatr Res 36: 187-193).

Pharmaceutical compositions of the invention [0055] The invention includes pharmaceutical compositions for use in the treatment of lysosomal storage diseases by providing spinal administration of defective or missing enzymes in such lysosomal storage disorders, thereby providing to replace the defective or missing enzyme in the brain tissues of the subject being treated. Delivery to target brain tissues occurs via the spinal route of administration. These methods effectively eliminate or reduce storage granules in the brain tissues of the subject being treated.

[0056] Lysosomal storage diseases that can be treated using the pharmaceutical compositions of the invention include, but are not limited to, Gaucher's disease (see, e.g., Barranger et al. Neurochemical Res. (1999) 24: 601-615 and NIH Technology Assessment Conference Statement , Feb. 27, 1995- Mar 1, 1995) including Types 1, 2 and 3, Fabry disease (see, eg, Takanaka et al. Exp. Hem. (1999) 27: 1149-1159, Ziegler et al Hum. Gene Ther. (1999) 10: 1667-1682 and Takanaka et al. Hum. Gene Ther. (1999) 10: 1931-1939), Tay-Sachs Disease (see e.g. Guidotti et al. Hum. Mol. Gen. (1999) 8: 831-838 and Daly et al. Proc. Natl. Acad Sci USA (1999 ) 96: 2296- 2300), Neimann-Pick disease, Types A, B and C, ornithine-δ-aminotransferase (OAT) deficiency (see, e.g., Jensen et al. Hum. Gene Ther. (1997) 8: 2125-2132 , Locrazza et al. Gene Ther. (1995) 2: 2228, Rivero et al. Hum. Gene Ther. (1994) 5: 701-707), hereditary homocysteinemia (see, e.g., McGill et al. Am. J. Med Gen. (1990) 36: 45-52, Eikelboom et al. Ann. Int. Med. (1999) 131: 363-365, Watanabe et al. Proc. Nat'l Acad Sci. USA (1995) 92: 1585-1589.), mannosidosis, fucosidosis, sialidosis, mucolipidosis, such as disease I-cells (mucolipidosis II) and pseudo-Hurler polidystrophy (mucolipidosis III), lipase acid deficiency such as Wolman's disease and storage cholesterol ester, sulfidide lipidosis including metachromatic dystrophy and multiple sulfatase deficit, MPS I (Hurler's disease) (see e.g. Lutzko et al. Hum. Gene Ther. (1999) 10: 1521-1532, Hartung et al. Hum. Gene Ther . (1999) 10: 2163-2172), MPS II (Hunter syndrome) (see, e.g., Rathmann et.

al. Am. J. Hum. Genet. (1996) 59: 1202-1209, Stronicek et al. Transfusion (1999) 39: 343350, Li et al. J. Med. Genet. (1999) 36: 21-27), MPS III (Sanfilippo syndrome) (see, e.g., Scott et al. Nat. Genet. (1995) 11: 465-467, Jone et al. J. Neuropath. Exp. Neur. ( 1997) 56 (10): 1158-1167), MPS IV (Morquio syndrome) (see e.g. Nothover et al. J. Inherit. Metab. Dis. (1996) 19: 357-365), MPS V (Scheie syndrome) (see, e.g., Dekaban et al. Arch. Pathol. Lab. Med (1976) 100: 231-245), MPS, VI (Maroteaux-Lamy syndrome) (see, e.g., Hershovitz et al.

J. Inherit. Metab. Dis. (1999) 22: 50- 62, Villani et al. Biochim. Biophys. Acta. (1999) 1453: 185-192, Yogalingam et al. Biochim. Biophys. Acta. (1999) 1453: 284-296), and MPS VII (Sly's syndrome) (see, e.g., Watson et al. Gene Ther. (1998) 5: 1642-1649, Elliger et al. Gene Ther. (1999) 6 : 1175-1178, Stein et al. J. Virol. (1999) 73 (4): 3424-3429, Daly et al. PNAS (1999) 96: 2296- 2300, Daly et al. Hum. Gene Ther. (1999) ) 10: 85- 94); and Sandhoff's disease.

[0057] A thorough review of the genetic etiology, clinical symptoms, and molecular biology of lysosomal storage diseases is provided in Scriver et al., Eds., The Metabolic and Molecular Basis of Inherited Disease, 7th Ed., Vol. II, McGraw Hill, (1995 ). Thus, enzymes deficient in the above diseases are known to those skilled in the art, some of them are listed in the Table below:

a lysosomal storage disease protein deficiency mucopolysaccharidosis type I mucopolysaccharidosis type II Hunter syndrome mucopolysaccharidosis type IIIA Sanfilippo Syndrome MPS type IIIB assembly Sanfilippoo a lysosomal storage disease MPS type IIIC Sanfilippo Syndrome mucopolysaccharidosis type IIID Sanfilippo Syndrome MPS type IVA Morquio Syndrome MPS type IVB Morquio Syndrome mucopolysaccharidosis type VI, mucopolysaccharidosis type VII syndrome Sly's mucopolysaccharidosis type IX

L-iduronidase iduronato-2-sulfatase heparan-N-sulfatase α-NA-cetylglucosaminidase protein acetylKoA deficiency: N-acetylglucosamino N-acetyltransferase 6-galactose 6-sulfatase 6-sulfatase β-galactosidase

N-acetylgalactosamine 4sulfatase β-glueuronidase hyaluronoglucosaminidase aspartylglucosaminuria

Cholesterol ester storage disease / Wolman's disease

Cystinosis

Danon's disease Fabry disease

Farber lipogranulomatosis / Farber disease fucosidosis galactosialidosis type I / II Gaucher disease types I / IIIII Gaucher disease globoid leukodystrophy / Krabbe's disease type II glycogenosis / Pompe disease GM1-gangliosidosis type I / II / III GM2-gangliosidosis type I / Tay's disease Sachsa GM2 type II gangliosidosis Sandhoff's disease GM2-lysosomal gangliosidosis storage disease α-mannosidosis type I / II β-mannosidosis metachromatic leukodystrophy metachromatic leukodystrophy mucolipidosis type I / sialidosis type I / II mucolipidosis type II / cell disease III I aspartylglucosaminidase Acid lipase cystine transporter Lamp-2 α-galactosidase A acid ceramidase α-L-fucosidase Protective protein

Glucocerebrosidase (β-glucosidase) galactocerebrosidase. Α-glucosidase.

Phosphotransferase mucolipidosis type IIIC polydystrophy pseudo-phosphotransferase γ-Hurler subunit multiple sulfatase deficiency multiple sulfonase ceroidolipofuscinosis neuronal, Batten's disease Palmitoyl thioesterase

CLN1 neuronal ceroidolipofuscinosis, Batten's disease tripeptidyl peptidase I

CLN2

Niemann-Pick type A / B disease Niemann-Pick acid sphingomyelinase disease Niemann-Pick type disease Niemann-Pick type disease C1 Cholesterol trafficking Niemann-Pick disease Niemann-Pick type C2 disease Cholesterol trafficking Niemann-Pick type pycnodysostosis cathepsin K type Schindler's disease I / II -galactosidase B sialic acid storage disease sialic acid transporter [0058] Thus, lysosomal storage diseases, which can be treated or prevented using the pharmaceutical compositions of the invention include, but are not limited to, mucopolysaccharidosis I (MPSI), MPS II, MPS IIIA, MPS IIIB, metachromatic leukodystrophy (MLD), Krabbe disease, Pompe disease, ceroidolipofuscinosis, Taya - Sachs, Niemann-Pick A and B and other lysosomal diseases listed above. In particularly preferred embodiments, the enzyme is a lysosomal storage enzyme such as α-L-iduronidase, iduronat-2-sulfatase, heparano N-sulfatase, α-N-acetylglucosaminidase, arylsulfatase A, galactosylceramidase, acid alpha-glucosidase, tripeptidyl peptidase hexosaminidase alpha, acid sphingomyelinase, α-galactosidase or any other lysosomal storage enzyme.

[0059] In even more preferred embodiments, the disease to be treated is MPS I and the enzyme being replaced is iduronidase. Those skilled in the art are aware of compositions containing iduronidase, see for example US Patent No. 6,585,971; U.S. Patent 6, 569, 661; U.S. Patent 6,524, 835; U.S. Patent 6,426,208; 6, 238, 662; U.S. Patent 6,149,909. Each of the patents mentioned above provides instructions on iduronidase compositions that can be used in the pharmaceutical compositions of the invention. Iduronidase is also commercially available as ALDURAZYME ™. Iduronidase can be a naturally occurring iduronidase that has been isolated from an animal source or, alternatively, it can be a surrogate iduronidase as produced according to the exemplary methods described in the above-mentioned patents. In certain embodiments, iduronidase can be produced surrogate in mammalian cells (eg, as described in the above patents) or plant cells (eg, as described in US 5, 929, 304.) [0060] In preferred embodiments, the pharmaceutical compositions of the invention reduce lysosomal storage granules in the meningeal and / or neuronal tissue of a subject exhibiting lysosomal storage disease. In a sense, therefore, the invention encompasses pharmaceutical compositions for use in reducing the size of the meningeal and / or neuronal tissue of a subject having a lysosomal storage disease by administering to the subject a pharmaceutical composition comprising an enzyme that is deficient in the lysosomal storage disease. In other embodiments, the invention also relates to pharmaceutical compositions for use in reducing a lysosomal storage disease of hypertensive hydrocephalus in a subject by providing the spinal subject with a pharmaceutical composition containing the enzyme deficient in lysosomal storage disease. Preferably the enzyme is idumnidase. The therapeutically effective amount of iduronidase in these contexts is any amount of iduronidase that produces a detectable decrease in lysosomal storage granules, reduces meningeal and / or neuronal mass, reduces the PRM associated edema present in the tires of an individual suffering from endocrine related lysosomal disorder and the like. Methods to determine if a subject's tires are swollen are well known to those of skill in the art and may include, for example, CAT scans.

[0061] In particular embodiments, the intrathecal administration discussed herein is used to treat symptoms that result from lysosomal storage granules in neuronal, glial or other brain tissues of an animal. These storage granules manifest a delay and / or regression in the development of the subject suffering from the disease. These symptoms and their alleviation by the treatment methods contemplated herein may be clinically assessed, for example using the Bayley scale of child development II, which includes monitoring the motor and developmental quotient. Development can also be assessed by language monitoring or other intellectual and motor development. Evoked potential studies, such as auditory or other evoked potential studies, can also be used to evaluate the effect of therapy on developmental delay and / or regression.

[0062] Other embodiments of the invention contemplate pharmaceutical compositions for use in the treatment of high pressure hydrocephalus caused by the presence of storage granules in the meninges near the arachnoid granules.

Such treatment can be monitored and evaluated using art-recognized methods for determining PMR pressure via lumbar puncture and / or intraventricular catheter. Any release or reduction of PMR pressure as a result of the therapeutic regimens of the invention will be considered a therapeutic benefit of the invention.

[0063] Treatment with the pharmaceutical compositions of the invention is also for use in alleviating the effects of lysosomal storage in the cervical meninges near the spinal cord at C1-C5 height or elsewhere along the spinal cord. Such storage results in symptoms associated with high CSF pressure as well as other symptoms associated with spinal cord compression. Storage results in progressive compression of the spinal cord with weakness of the lower limb, loss of bowel and bladder control and sensory deficits. Such symptoms can be monitored, for example, using a neurological examination of Babinski's abnormal reflexes, deep tendon reflexes, motor function or sensation. Neurophysiological deficits resulting from spinal cord compression can be assessed using somatosensory evoked potentials. Alternatively, magnetic resonance imaging with or without contrast agent can be used to identify the anatomical location of the compression as well as to assess edema or other signs of spinal injury at the site of compression. The high pressure exerted by PMR will lead to physiological manifestations such as headache, swelling and the like. Any reduction in pressure exerted by PMR, reduction of edema or any improvement in neurophysiological deficiencies, tendon reflexes, motor functions or sensation observed as a result of administering a therapeutic regimen will be considered a therapeutically beneficial effect. The subject may be particularly monitored for any level of improvement in lower limb weakness, bowel and bladder control, and sensory deficiency associated with spinal cord compression.

[0064] Perivascular storage of lysosomal storage granules around brain vessels can cause cysts. Such cysts and the efficacy of the therapeutic regimens of this application against such cysts can also be evaluated using MRI scans to determine the size and number of such cysts. Any reduction in the size and / or number of cysts will be considered a therapeutically beneficial effect in the methods of the invention.

[0065] Any release or reduction of PMR pressure, reduction in the size and / or number of cysts, or any other reduction in symptoms due to the presence of lysosomal storage granules as a result of the therapeutic regimens of the invention will be considered a therapeutic benefit of the invention. Such decreases are preferably on the order of at least 5% compared to levels of such symptoms before administration. Of course, a larger decrease, e.g. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or more would be more favorable. Most preferably, the symptoms are reduced / alleviated to such an extent that the subject's symptoms are indistinguishable from the same signs observed in a normal healthy subject, of similar sex, age, and physical characteristics.

Evaluation of methods on model animals [0066] These methods can be evaluated using lysosomal storage disease models that are known to those skilled in the art. For example, the psi model MPS I can be used as described in the examples below. Other MPS models can also be used. Many preclinical studies are based on mouse models for a given disease. One such model is the MPS model described at least 1 example of US Patent 6582692, whose details of crossing the Gusmps / + mice in the original background C57BL / 6 (B6) (Jackson Laboratory, Bar Harbor, ME., USA) with the congenic B6Gusa strain (Pfister et al. (1982) Biochem Genet 20: 519-535) to produce Gusmps / a progeny for breeding colonies in which both parents were always Gusmps / a and the offspring carried mps / a, a / a or mps / mps allele combinations. Parameters for GUS activity can be monitored as described in this patent.

[0067] Still another model that may be useful for evaluating the methods of the invention is even illustrated in US Patent 6002067, which is a mouse transgenic model for iduronidase deficiency. Of course, those skilled in the art will be aware of other models that can be used to evaluate the methods of the invention. As soon as the methods have been evaluated on such model animals, the methods are then easily scaled and adapted for the treatment of other mammalian subjects, such as primates and preferably human subjects.

[0068] One of the most dramatic features of MPS disease is the appearance of large cytoplasmic storage vacuoles in microscopic tissue sections (Vogler et al. (1990) Am J Pathol 136: 207-217). In one example, using the MPS mouse model, MPS mice were spinal injected with a composition containing either iduronidase or saline alone, are sacrificed 4 weeks after administration, and examined histologically as below. Mice are killed by cervical dislocation and immediately perfused through the left ventricle, first with saline solution and then with 10% neutral buffered formalin. Fixed animals with exposed viscera are then immersed in formalin before histopathological evaluation. Selected tissues are processed using routine techniques, embedded in paraffin, cut to approximately 5 microns, stained with hematoxylin and eosin and examined microscopically. In particular, it is desirable to perform such a histopathological analysis on the meningeal and / or neuronal cells of animals.

[0069] Brain sections from control MPS animals should have moderate to heavy, diffuse, cytoplasmic vacuoles in meninges and / or neuronal cells. Such cells from treated mice have significantly reduced storage vacuoles resulting in normal tissue architecture.

[0070] To assess the presence of storage granules in the brain, mice are sacrificed by cervical dislocation, brains are removed and one hemisphere is fixed in 10% inert buffered formalin. 5 mM sections are stained with hematoxylin and eosin (H i

E).

[0071] The animals discussed above, both neonates and adults, can be treated by spinal administration of iduronidase. Model mice, three-day neonatal mice and e.g. adult mice 7-13 weeks old at the time of injection can be treated

0.01 μg to about 5 mg of enzyme. Note that the dose is similar to the 1 / 1000th dose that is required for larger mammals such as dogs. For spinal administration to newborns, mice were anesthetized by inhalation of halothane, and iduronidase in 30 μl saline (with 2% dye added) can be injected between the sixth lumbar and second sacral vertebra using a 30-gauge needle. Successful introduction into the cerebrospinal fluid space is immediately detected as green streaks extending from the spine and diffusing towards the brain. For spinal administration to adults, MPS mice were anesthetized with avertin (tribromoethanol) and a 1 cm incision was made across the entire skin parallel to the spine to visualize the position of the individual vertebrae. Iduronidase with a 2% dye can then be injected between the last thoracic and second lumbar vertebrae.

[0072] More and more times after this treatment, mice are sacrificed and tissues are analyzed for iduronidase levels. The therapeutic levels of the iduronidase enzyme were evaluated as any level that produces detectable reductions in vacuole storage. Of course, the above model tests are presented by way of example only, with other example model tests described hereinafter in the Examples, these model tests can be easily modified without departing from the scope of the claimed invention.

Modification of the enzyme to facilitate improved uptake [0073] In the pharmaceutical compositions of the invention, it may be advantageous to ensure that the enzyme is administered to the subject by intrathecal administration that contains a residue that can be easily captured by the high affinity uptake on the surface of brain cells. For example, such a receptor may be a 6-mannose phosphate receptor and the enzyme contains up to about an average of at least 20% bisphosphorylated oligosaccharides per enzyme. In other embodiments, the enzyme may contain 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45% bis-phosphorylated oligosaccharides per enzyme. Although such bis-phosphorylated oligosaccharides may be naturally present on the enzyme, it should be noted that the enzymes can be modified for the presence of such oligosaccharides. For example, those skilled in the art are aware of enzymes that are capable of catalyzing the transfer of N-acetylglucosamine L-phosphate from UDPGlcNAc to the 6 'position of α-1,2-mannoses bound on lysosomal enzymes. Methods and compositions for the preparation and use of such enzymes are described, for example, in Canfield et al. in US Patent 6, 537, 785 and US Patent 6, 534, 300.

[0074] In other embodiments, the lysosomal enzymes for use in the invention can be conjugated to RAP and RAP polypeptides that selectively bind to LRP receptors that may be present on brain cells. As such, these RAP molecules will serve to increase the transport of lysosomal enzymes across the blood-brain barrier and / or to provide agents for cell lysosomes within the CNS. Methods and compositions for making enzyme compositions that contain RAP residues attached thereto and are described in detail in US Patent Application 10/206 448 filed July 25, 2002 and in US Patent Application 10/600 862 filed June 20, 2003.

[0075] In yet another embodiment, those skilled in the art may use the delivery of melanotransferrin-conjugated enzymes (p97), as described, for example, in US Patent 6,455,494 and US Patent 5,981,194. Of course, the above agents that increase the delivery and / or uptake of therapeutic agents into the brain tissue are only examples and those skilled in the art will be aware of other receptors, ligands or other agents that can be used in a similar context to deliver therapeutic agents across the brain-PMR border or even BBB.

Combination therapy to generate the subject's tolerance to enzyme replacement therapy [0076] It has been found that when drugs such as recombinant proteins and other therapeutic agents are administered, the subject may increase the immune response against these agents, resulting in the production of antibodies that bind and interfere with therapeutic action and also cause acute or chronic immune responses. This problem is most significant for therapists who are proteins because proteins are complex antigens and in many cases the subject has not been in contact with these antigens. Thus, in certain aspects of the invention that may be useful for rendering a subject receiving a therapeutically tolerant enzyme for enzyme replacement therapy. In this context, enzyme replacement therapy may be administered to the subject as a combination therapy with a tolerance regimen.

[0077] Co-owned, pending US Patent Application 10/141 668 discloses the treatment of lysosomal storage disorders using immune tolerance induction. Briefly, the use of such a tolerance-inducing regimen may be useful for preventing the subject's immune response to enzyme replacement therapy, and thereby reducing or otherwise rendering ineffective potential beneficial effects of enzyme replacement therapy.

[0078] In a preferred method, the method contemplates reducing or preventing a clinically significant antigen-specific immune response to recombinant human αL-iduronidase used to treat mucopolysaccharidosis I (MPS, I) where iduronidase is administered intrathecally. The method is used initially 30-60 days according to the T cell immunosuppressive regimen such as cyclosporin A (CsA) and antiproliferative agent such as azathioprine (Aza) in combination with weekly low-dose iduronidase spinal infusions. The typical strong IgG response to weekly iduronidase infusions is significantly reduced or stopped with the 60 day regimen of immunosuppressants, cyclosporin A (CsA) and azathioprine (Aza), in combination with weekly spinal low-dose rhIDU infusions. By using such tolerance regimens, it will be possible to cause subject tolerance to higher therapeutic doses of iduronidase for up to 6 months without increasing the antibody titer against iduronidase, or indeed any other enzyme that can be used to replace the enzyme in lysosomal storage disease. Such tolerance development schemes are described in US Patent Application 10/141 668.

Intrathecal administration of pharmaceutically acceptable formulations [0079] As discussed above, the invention is based on the surprising discovery of the therapeutic effectiveness of using intrathecal administration of enzyme replacement therapy for lysosomal storage disease. In one embodiment, the enzyme is administered by introducing the subject into the central nervous system, e.g., the subject's cerebrospinal fluid. In some aspects of the invention, the enzyme is intrathecally introduced, e.g. to the lumbar region or large reservoir or intraventricularly to the space of the cerebral ventricles.

[0080] Those skilled in the art are aware of the devices that can be used to perform spinal administration of a therapeutic composition. For example, treatment may be given using the Ommaya reservoir, commonly used for spinal administration of meningitis carcinomatosis (Lancet 2: 983-84, 1963). More precisely in this method, the chamber tube is introduced through a hole in the front corner and is connected to the Ommaya reservoir installed under the scalp and the reservoir subcutaneously pierced to the spinal delivery of the specific enzyme being replaced, which is injected into the reservoir. Other devices for intrathecal administration of therapeutic compositions to an individual are described in US Patent 6,217,552. Alternatively, the drug may be administered intrathecally, for example by a single injection or continuous infusion. It should be understood that the treatment may be dosed in the form of single or multiple doses.

[0081] The term "intrathecal administration" as used herein is intended to include delivering the pharmaceutical composition directly to the cerebrospinal fluid of the subject, by techniques, including lateral ventricular injection via a borehole or reservoir or lumbar puncture or the like (described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1: 169-179, the contents of which are incorporated herein by reference.) The term "lumbar region" is intended to include the area between the third and fourth lumbar vertebrae (lower back) of the vertebrae and, more inclusive, the L2-S1 region of the spine. The term "cisterna magna" is intended to include access to the space around and below the cerebellum through the opening between the skull and the top of the spine. The term "cerebral ventricle" is intended to include cavities in the brain that are continuous from the central spinal canal. Administration of the pharmaceutical compositions of the invention to any of the aforementioned sites may be accomplished by direct injection of the composition or by the use of infusion pumps. For injection, the composition of the invention may be formulated in liquid solutions preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or phosphate buffer. In addition, the enzyme may be formulated in solid form and re-dissolved or suspended immediately prior to use. Freeze-dried forms are also included. The injection can be, for example, in the form of a bolus injection or continuous infusion (e.g. using an infusion pump) of the enzyme.

[0082] In one embodiment of the invention, the enzyme is administered by injection into the lateral ventricle of the subject. The injection can be done even with the help of a drilled hole made in the subject's skull. In another embodiment, the enzyme and / or other pharmaceutical formulation is administered through a shunt surgically inserted into the subject's cerebral ventricle. For example, the injection can be performed into the lateral chambers, which are larger, even if the injection into the third and fourth smaller chambers could be performed.

[0083] In yet another embodiment, the pharmaceutical compositions used in the invention are administered by injection into the cisterna magna or lumbar region of the subject.

[0084] In another embodiment of the method of the invention, the pharmaceutically acceptable formulation provides sustained delivery, for example "slow release" of the enzyme or other pharmaceutical composition used in the invention at the subject for at least one, two, three, four weeks or longer periods after a pharmaceutically acceptable the formulation is given to the subject.

[0085] The term "extended delivery" as used herein is intended to mean continuous delivery of the pharmaceutical composition of the invention in vivo over a period of time after administration, preferably at least a few days, a week or even several weeks. Prolonged delivery of the composition can be manifested, for example, by a further continuous therapeutic effect of the enzyme over time (e.g., prolonged delivery of the enzymes can be manifested by a continuous reduced amount of storage granules in a subject). Alternatively, extended enzyme delivery may be manifested by detection of the presence of the enzyme in vivo over time.

[0086] The pharmaceutical formulation used in the invention contains a therapeutically effective amount of enzymes for use in enzyme replacement therapy in lysosomal storage disease. Such a therapeutically effective amount is any effective amount, in doses and for periods of time, to achieve the desired result. In preferred embodiments, the compositions contain a therapeutically effective amount of iduronidase. The therapeutically effective amount of iduronidase may vary depending on factors such as the condition of the subject, age and weight of the subject, and the ability of the enzyme (alone or in combination with one or more other agents) to elicit the desired response in the subject. Dosage regimens can be adjusted to achieve the optimal therapeutic response. A therapeutically effective amount is also one in which any toxic or harmful effects of the compositions are balanced by therapeutically beneficial effects. The non-limiting range for a therapeutically effective concentration of iduronidase is 0.001 μg enzyme / ml to about 150 μg enzyme / ml. It should be noted that dose values ​​may vary depending on the severity of the condition to be alleviated. It should also be understood that for any particular subject, specific dosage regimens should be adjusted over time, according to the individual needs and professional judgment of the person administering or supervising the administration of enzyme replacement therapy, and that the dosage ranges provided herein are exemplary only and are not intended to limit the range or practice of the claimed invention.

[0087] The enzyme composition is preferably in the form of a unit dose for injection. Examples of carriers or diluents suitable for preparing such injectable doses include diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol of sorbitan and polyoxyethylene sorbitan fatty acid esters, pH adjusting agents or buffers such as citrate sodium, sodium acetate and sodium phosphate, stabilizers such as sodium metabisulfite, EDTA, thioglycolic acid and thiomylic acid, isotonic agents such as sodium chloride, glucose, local anesthetics such as procaine hydrogen chloride and lidocaine hydrochloride. In addition, ordinary solubilizing and analgesic agents may be added. Injections can be made by adding such carriers to the enzyme or other active substances by following procedures well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0088] Pharmaceutically acceptable formulations can be easily suspended in aqueous vehicles and introduced using conventional hypodermic needles or using infusion pumps. The formulations may be sterilized, preferably, by gamma radiation or electron beam sterilization prior to introduction.

Kits [0089] The agents used may be provided in the form of a kit, which kit may further include instructions for use. Such a kit will contain enzymes for use in the treatment of lysosomal storage disease, usually in a dose and form suitable for administration to the host. The kit will usually include a device for delivering the enzyme intrathecal The kit may further comprise an T cell immunosuppressant agent, in a form suitable for administration, and may further contain test reagents for monitoring blood levels of the agent and / or to determine inhibition of T cell activity. The antiproliferative agent may also be in a form suitable for administration.

[0090] The kit may also be provided for coupling an antigen, especially a polypeptide antigen, to high uptake of the residue to produce a tolerogenic composition. For example, a residue such as phosphate mannose or coupled to a linker suitable for linking sugars and polypeptides, as described above, may be provided. The rest of the high uptake can also be delivered in unconjugated form, in combination with a suitable connector and instructions for use.

[0091] Another kit may contain instructions for intrathecal administration of the therapeutic compositions of the invention, in addition to the therapeutic compositions. In certain embodiments, kits of the invention may include a catheter or other devices for intrathecal administration of enzyme replacement therapy, which are pre-loaded therapeutic compositions of the invention. Even catheters with 0.001 mg 0.005 mg 0.01 mg 0.015 mg 0.02 mg 0.03 mg 0.04 mg 0.05 mg 0.06 mg 0.07 mg 0.08 mg, 0.09 mg, 0, 1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg or 1.0 mg or greater iduronidase in a pharmaceutically acceptable formulation are explicitly contemplated. Other enzymes for use in lysosomal storage diseases may also be similar in factory intrathecal administration catheters. Exemplary catheters may be disposable catheters that can be discarded after use. Alternatively, pre-installed catheters may be refilled and presented in kits that have the appropriate amount of enzyme to replenish such catheters.

[0092] Additional aspects and details of the invention will be apparent from the following examples, which are intended as illustrative rather than limiting.

EXAMPLE 1

Protocols for assessing direct brain injection by recombinant human indruidase [0093] Those skilled in the art are also aware of well-known canine models for lysosomal storage diseases. In one embodiment, dogs with MPS I are used to evaluate the effectiveness of the methods of the invention. For such assays, it is desirable that normal dogs and dogs with MPS, and be evaluated simultaneously. The following example provides at least examples of protocols to be used in conjunction with the methods described herein.

[0094] To assess the entry of enzymes into the brain of normal dogs, normal Beagle dogs (at least 2 initially planned to 4 possible) are prepared for anesthesia and sterility. The Ommaya reservoir or equivalent device is implanted with a catheter chamber located in the lateral chamber. The PMR reservoir and lumbar catheter can also be implanted in the lumbar region. PMR is taken to confirm patency. Enzyme administration and PMR sampling is carried out in a lateral chamber in one of the beagle dogs. Enzyme and PRM specimens are administered in the lateral ventricle in one of the beagle dogs. The arrangement in the lumbar region of this beagle dog serves as a backup in the event of irreversible problems occurring in the original access site. A second beagle is prepared to obtain the enzyme and PRM samples in the lumbar region. The arrangement in the lateral ventricle of this second beagle will serve as a backup in the event of irreversible problems occurring in the original access site. The enzyme is given by weekly injections for four weeks. Pharmacokinetic studies of PRM clearance are evaluated using a set of time samples in the first and last week of injection. All PRM samples obtained are analyzed for safety, pharmacokinetics and pharmacodynamics of enzyme penetration. If complications arise when placing the ventricular system in normal beagle dogs, the methods of the invention may be evaluated using a delivery system located only in the lumbar region. Thus, administration of the enzyme and PRM samples will only occur in the lumbar areas. At the end of treatment, brain tissue may be harvested to assess iduronidase activity using validated analysis. Brain tissues will also be analyzed for storage using light microscopy and confocal immunofluorescence. Tissues both proximal and distal to the ventricular permeation site can be evaluated for enzyme permeation.

[0095] To determine enzyme entry in model animals for lysosomal storage disease, the above protocol is repeated using dogs with MPS I.

[0096] To evaluate the response of animals to treatment, many parameters can be controlled. To obtain a baseline assessment, it may be desirable to conduct a clinical trial to assess physical condition, signs of life and weight. This assessment should preferably be supplemented by clinical laboratory analyzes for the determination of blood count (CBC), biochemical profile and urine analysis of animals. Urine samples should be analyzed for the presence of glycosaminoglycans, serum analysis should be performed to assess the presence of anti-iduronidase antibodies, as they may affect the amount of iduronidase that should be administered. The plasma iduronidase activity should also be assessed. Baseline assessment should also include PRM analysis for standard PRM laboratory analysis (cell number, protein, glucose and cytology), GAG, ELISA for anti-id antibodies and iduronidase analysis. Cells present in PRM should be evaluated for granularity using simple staining. These parameters should then be evaluated periodically throughout the treatment period. At the end of the analysis period, brain tissues can be taken and further analyzed. Such analyzes may include brain biopsy for performing iduronidase and GAG tissue levels analysis. The pathology of animals with MPS I can be assessed using light and electron microscopy and confocal immunofluorescence can also be performed using anti-iduronidase antibodies.

[0097] The following is a discussion of the general methods used to perform the assessments outlined above.

[0098] Clinical study: To assess the physical condition of the animals, the general physical examination should record posture, activity, behavior and general appearance, preferably daily throughout the duration of the experiment, the study should record the animal's life signs (heart rate, body temperature and respiratory rate), especially after each injection. Height can be assessed periodically by body weight measurements.

[0099] Levels of PRM and plasma α-L-iduronidase: Enzyme levels can be measured in plasma and PRM just prior to administration of the enzyme to PRM weekly. PRM is obtained after sterile preparation of the PMR port and access is made with a sterile needle. The enzyme in blood samples is stabilized by adding 1/10 volume of 100mM NaPO4 / citrate pH

4.0. The enzyme is analyzed for iduronidase using validated analysis with an artificial 4-methylumbelliferyl-α-1-iduronide substrate. Network fluorescence is determined by fluorometry at 365 nm excitation and 440 nm emissions. One iduronidase unit corresponds to the number of micromoles of substrate split per minute at 37 ° C under analysis conditions.

[0100] α-L-brain iduronidase: Enzyme levels can be measured in biopsy specimens. In dogs with MPS I biopsies may be obtained prior to perfusion. The brain sample is frozen in a labeled vial in liquid nitrogen. After thawing, the sample is weighed quickly and 3 volumes PAD (10 mM phosphate buffer pH 5.8, 0.02% azide and 0.1 mM dithiothreitol) + 0.1% Triton X-100. The tissue sample is ground in a Dounce glass homogenizer for a minimum of 10 strokes, while on ice, and the homogenate is cleaned of large particles by centrifugation in a microcentrifuge for a few seconds. The extract is stored by freezing. The enzyme is analyzed for iduronidase using a validated analysis with an artificial 4-methylumbelliferyl-α-1-iduronide substrate. Pilot analyzes should preferably be carried out to determine the time of analysis and whether dilution is necessary. Network fluorescence is determined by fluorometry at 365 nm excitation and 440 nm emissions. One iduronidase unit corresponds to the number of micromoles of substrate split per minute at 37 ° C under analysis conditions.

[0101] GAG analyzes: At the end of therapy, dogs may be euthanized, and brain tissue samples taken by biopsy and quickly frozen with liquid nitrogen for subsequent tissue analysis of glycosaminoglycan. For GAG tissue analysis, sulfated glycosaminoglycans will be analyzed using a modification of the Bjomsson Alliance Blue method as published (Kakkis et al., Biochem Mol Med. 1996; 58 (2): 156-67). The amounts of GAG can be determined by comparing to dermatan sulfate standards. Urinary and PMR quantification of GAG is completed by a method almost identical to that used to quantify the amount of tissue GAG ​​content performed on urine and PMR samples.

[0102] PMR storage: Cellular debris from PRM can be determined using simple staining and cells readily scored for GAG storage.

[0103] PMR Pharmacokinetic Studies: Pharmacokinetic studies can be completed on each treated dog during the first and last week of enzyme replacement therapy to control clearance of α-L-iduronidase from PMR after infusion. After enzyme administration to PMR via the ventricular port, samples are taken from the same enzyme administration site in 1, 2, 4 hours. Samples are taken and prepared as in the PMR sample section. Data are coupled as time versus PMR iduronidase activity. The half-life of iduronidase in the circulation can be determined.

[0104] CBC, biochemical profile and urinalysis: Blood samples are taken every two weeks for CBC and biochemical profile. Urine analysis with reagent strips is also carried out every other week on a fresh urine sample to control elements such as proteinuria and hematuria.

[0105] ELISA for specific-L-iduronidase antibodies: Serum samples are taken and frozen at -20 ° C for subsequent antibody analysis. Iduronidase specific antibodies are detected by a standard ELISA protocol using goat anti-dog IgG labeled with alkaline phosphatase as a secondary antibody. Antibodies in PMR are determined by the same method, although it is expected that a small dilution may be needed.

[0106] Supplied enzyme composition: Recombinant human α-L-iduronidase is provided by BioMarin Pharmaceutical from major kits that may or may not be approved for human use. The enzyme should preferably meet all relevant specifications required for enzyme therapy and safe administration including passage potential, activity, sterility and endotoxin levels. The dosage form consists of the enzyme at 100,000 u / ml in formulation buffer (100 mM NaPO4, pH 5.8, 150 mM NaCl at pH 5.3-5.8).

[0107] Permanent placement of a ventricular device: Procedures have been described for sampling through a ventricular system (McCully et al; Poplack et al; Moir and Dow et al; Kusumi and Plouffe; HASLBERGER and Gaab). Some of these also include brain injuries and do not allow precise positioning of the delivery system. We will use a technique that allows the researcher to obtain multiple sterile PMR samples or to administer multiple PMR injections to non-anesthetized animals that are tamed with the minimum dose of sedatives. The procedure involves placing a permanent catheter into the lateral ventricles as well as into the spinal space of the lumbar region of the spine.

[0108] In the examples discussed here, animals, even two normal, male, adult, laboratory, Beagle-bred dogs and two male dogs with Mucopolysaccharidosis I are used. Dogs are given atropine (0.045 mg / kg), intravenously Propofol (1-6 mg / kg) to increase the effect. The dogs were intubated and kept under anesthesia with isoflurane / oxygen and placed on a heated pad during surgery to maintain body temperature. Normal saline will be administered to retain fluid. Antibiotics may be given before and during surgery to prevent infection.

[0109] Dogs are placed on the abdomen with the head supported to ensure that the airways remain unobstructed. The occiput and dorsal midline are surgically cut and wrapped. A sterile technique and magnifying glass are used throughout the procedure. The appropriate catheter length is predetermined by measuring the thickness of the first two cervical vertebrae (C1 and C2), the distance from C2 and the distance to the reservoir. The volume of fluid necessary to top up the volume of the catheter and reservoir (dead space) is calculated.

[0110] The skin is cut in the median line from the occipital heigth along the dorsal median line to expose the grand opening, the junction of C1 and the occiput, and the apoptotic membrane. The subcutaneous muscles are sharply dissected and the cervical ligament is separated. Using a pneumatic drill and a scalpel, a small hole is formed in the rear elements (?) [Posteruir ekenebts] C1 and a 2mm horizontal cut is made in the dura for entering the large tank. Using a surgical hook, the previously measured length of the perforated Spetzler lumbar catheter containing the dagger is sutured to the ventricular space and PMR is taken to confirm patency. The catheter is anchored to the muscle near the reservoir. Hemostasis is carried out by means of a bipolar electrocautery unit, a subcutaneous subdural pocket is created in the occipital area to accommodate the Ommaya reservoir. The reservoir is secured with a non-absorbable suture to the occipital periosteal skull. The remaining outer part of the catheter is stretched into the subcutaneous pocket, and a metal step-down connector is used to attach the catheter to the Ommaya reservoir and a silk suture can be used to secure the connection.

[0111] To determine the patency of the catheter, a small amount of PMR that only exceeds the total dead space of the catheter and reservoir is gently withdrawn using a 5/8-inch needle attached to a 1 cc syringe. The reservoir is secured with sutures not absorbable to the occipital periosteal skull. The system is examined for leakage, and the surgical site is enclosed in anatomical layers with intermittent 3.0 Vicryl sutures. The skin is then closed with nylon sutures.

[0112] PMR collection or reservoir injections are performed using a sterile technique (surgical skin scraping and sterile gloves) with a 5/8 inch needle attached to a 1 cc syringe. PMR should be collected gently and evenly.

[0113] Postoperatively, the dog is monitored and intravenous fluids are administered as needed until he is able to stand, eat and drink. The analgesic buprenorphine (0.01 mg / kg SQ over 12 hours) may be given if necessary to relieve discomfort. Antibiotics can be administered postoperatively for 10 days. The dog should be clinically examined daily.

[0114] To maintain patency, the system should be washed weekly. This will allow PMR sampling and enzyme administration. For enzyme administration or PMR collection, dogs are tamed with 0.1 mg / kg of acepromazine or more as needed. The skin above the reservoir is strapped and surgically scraped. The location of the reservoir behind the ear is determined by wearing sterile gloves and is entered using a 25-gauge needle attached to a 1 ml syringe. Special care should be taken when entering the tank dome a few millimeters from the skin puncture area to reduce exogenous contamination. A volume of PMR equal to the catheter volume plus the reservoir is removed and discarded by removing the syringe and discharging its contents; then the desired amount of PMR is withdrawn or the enzyme is fed into the reservoir. When the enzyme (18,000 units / mL, PMR) is administered, it is preferable to "chase" the enzyme with a volume of saline equivalent to the volume of the dead space of the catheter and reservoir. This ensures that the enzyme is administered directly to the ventricular system. PMR sampling and enzyme administration continue as discussed above after catheter placement.

[0115] For tissue analysis, after eight weeks of enzyme treatment, dogs are deeply anesthetized (loss of pinching reflex to the finger and eyelid reflexes) with an excessive dose of sodium pentobarbital and rinsed with cardiac heparinized saline. A small hole is made in the skull in the frontal area and a small section of the brain is removed. The dog is then subjected to cardiac perfusion with 4% paraformaldehyde.

[0116] Throughout the above treatment protocols dogs should be closely monitored for signs of anaphylactic reaction during and immediately after administration of the enzyme. Signs of reaction may include changes in behavior such as anxiety, irritability or extreme stillness, as well as vomiting, bowel movement and loss of color of the mucous membranes. If any of these symptoms or other undesirable effects occur, administration should be stopped, diphenhydramine may be given followed by saline drip and oxygen administration. The infusion can be continued when the reaction stops.

[0117] Dogs are also monitored for infection due to the emergent catheter and are treated with appropriate means (catheter removal, local infection control, systemic antibiotics). If there is an infection, such as ventricular inflammation, enzyme treatment will be suspended until adequate treatment for the gentamicin infection.

[0118] The following examples describe the results of studies conducted on the intrathecal administration of iduronidase to MPS I animals using some or all of the methods described in the above example.

EXAMPLE 2

Enzyme Administered By Intraventricular Injection Penetrates Blood Brain Barrier And Is Detected In Brain Tissue [0119] Administration of enzymes directly to the lysosomal storage site induces brain damage in subjects with lysosomal storage disease has been difficult to date. The large enzyme complexes necessary to treat these diseases typically cannot penetrate the blood-brain barrier. To determine the effective mode of transmission of these enzymes across the brain-PMR border, two routes of enzyme administration were studied in the rat and canine model of MPS lysosomal storage disorder I.

[0120] For intraventricular administration of the enzyme, rats were injected into the lateral ventricle using sterotactic guidance, or 5-10 ml of recombinant human iduronidase (rhIDU) or control protein. Animals were sacrificed 24 hours after injection and obtaining brain sections.

[0121] Brain sections were analyzed for the presence of rhIDU using confocal microscopy with anti-iduronidase antibodies. Immunohistochemical analysis showed that the injected enzyme is captured by brain neurons and, furthermore, that iduronidase is located in lysosomes in neuronal cells. Anti-IDU staining indicates that the enzyme penetrates the brain tissue a few millimeters, but there is a decreasing enzyme gradient, which means that the further from the injection site, the less enzyme is detected in the brain. Staining also indicates that the enzyme half-life was about 7 days.

[0122] Brain sections were also analyzed for rhIDU activity.

EXAMPLE 3

Enzyme Administered by Spinal Injection Penetrates and Is Detected in Brain Tissue [0123] To determine whether spinal injection of an enzyme involved in lysosomal storage disorder can penetrate the blood-brain barrier as effectively or more effectively than intraventricular injection, spinal injection was performed on PMR in dog subjects .

[0124] Animals (n = 2 / group) were administered 1 cc rh-iduronidase, with a total protein content of 0.33 mg, 1 mg, or 3 mg, by injection into a large reservoir. This protocol was repeated weekly for a total of four weeks. Brain sections were collected for analysis 48 hours after the last injection. For analysis, the right hemisphere of the brain was cut into slices by the head and alternative sections were analyzed for enzyme activity, immunohistochemical location of the enzyme in the brain and glycosaminoglycan content in the brain sections. The left hemisphere of the brain was cut into coronary patches and analyzed microscopically and electron microscopy.

[0125] Analysis of brain enzyme levels in subjects after spinal injection (Figure 1) shows that animals given 0.33 mg ofiduronidase have a 5-fold increase in enzyme in the brain compared to control animals (mean, 65 ± 28 units / mg protein), animals that received 1 mg / injection had a 7-fold increase in enzyme (mean enzyme levels of 89 ± 62 U / mg), while animals receiving 3 mg enzyme / injection had a 17-fold increase in enzyme levels, with an average iduronidase level of approximately 224 ± 32 U / mg. Thus, increasing the dose of iduronidase administered to a subject increases the level of iduronidase detected in the brain.

[0126] In similar experiments, 6 dogs were treated with low (0.46 mg / injection), medium (1.08 / 1.38mg / injection) and high (4.14 mg / injection) doses of rh-iduronidase administered by large reservoir once weekly for four weeks and brain iduronidase content analyzed 48 hours after the last dose (see Table 1 for individual doses). Two spinal treated dogs at each dose level were compared to iduronidase enzyme levels in two untreated normal dogs. IT-treated dogs had 5.6, 7.5 and 18.9-fold enzyme levels of untreated or vehicle-treated animals at low, medium and high doses, respectively (Table 1). If the corrective enzyme concentration is given as little as 2-5% of the normal, these levels represent concentrations much higher than the required corrective enzyme concentrations.

Table 1: RhIDU dose-response actions administered to normal IT dogs

<td>Dose weekly IT rhIDU (mg)</td><td>Whole brain^¥</td><td>multiplicity normal</td><td>Brain superficial^¥</td><td>multiplicity normal</td><td>Brain deep^¥</td><td>multiplicity normal</td>

<td>untreated / treated placebo Normal</td><td> 11,9 ± 1,95 [10,1 15,0]</td><td> 1</td><td>NW</td><td> 1</td><td>NW</td><td> 1</td>

<td>Low (0.46)</td><td> 66,4 ± 4,07</td><td>^5,6 p =</td><td> 101 ± 19,5</td><td>8.5 p =</td><td> 31,8 ± 3,75</td><td>2.7 p =</td>

<td></td><td> [63,5, 69,3]</td><td> 0,0001*</td><td> [87,5, 115]</td><td> 0,0001*</td><td> [29,1, 34,4]</td><td> 0,0002*</td>

<td>Medium</td><td> 89,0 ± 18,2</td><td>7.5 p =</td><td> 121 ± 43,4</td><td>10.2 p =</td><td> 52,3 ± 0,64</td><td>4.4 p &lt;</td>

<td> (1,08/1,38)</td><td> [76,0, 102]</td><td> 0,0001*</td><td> [90,5, 152]</td><td> 0,0011*</td><td> [51,8, 52,7]</td><td> 0,0001*</td>

<td>High</td><td> 225 ± 89,5</td><td>18.9 p =</td><td> 355± 198</td><td>29.8 p =</td><td> 70,6 ± 14,1</td><td>5.9 p =</td>

<td> (4,14)</td><td> [161, 288]</td><td> 0,0014*</td><td> [214, 495]</td><td> 0,0057*</td><td> [60,6, 80,5]</td><td> 0,0001*</td>

Iduronidase levels are calculated from the average values ​​for each region for each dedicated dog. Mean means for each animal are ± standard deviation. N = 5 for the untreated group and N = 2 for each dose group. Id Iduronidase levels are expressed in units of iduronidase per mg of protein. * Statistically significant. NW means not done.

[0127] Rh-induronidase levels were also measured in deep brain and surface brain tissue (Figure 2) from animals treated with or 0.33 mg, 1 mg or 3 mg enzyme. Again, analysis showed that a higher dose of the enzyme with an increased amount of iduronidase detected in brain tissue from the 3 mg / injection group demonstrating a 5-fold increase in deep brain tissues. Iduronidase measured on the surface of the brain tissue was detected in an 8-fold difference in animals receiving 0.33 mg protein / injection, while animals receiving 3 mg / injection showed a 27-fold increase in surface expression of iduronidase compared to the control group. Thus, while the majority of iduronidase detected in surface brain tissue, a significant amount penetrates deep brain tissues, indicating that this type of treatment would be useful for the administration of enzymes in lysosomal storage disorders of deep brain tissues. Additional experiments where low, medium and high doses were 0.46 mg; 1.08 / 1.38 mg and 4.14 mg; respectively, showed that the deep brain had 2.7 specimens,

4.4 and 5.9 times normal effects at these respective doses.

[0128] Immunohistochemical confocal microscopy analysis showed that large amounts of Rh iduronidase can be detected on the surface of the cortex and inside the hippocampus cells. In particular, glial cells in the hippocampus, a part of the brain involved in memory, take significant amounts of the enzyme. Staining also showed that the enzyme diffuses into the brain and some glial cells stain brightly with anti-iduronidase. Higher doses do not cause significantly higher α-L-iduronidase activity in deep brain areas, and hence a dose of approximately 1 mg was selected for the treatment of dogs with MPS I in further studies.

[0129] These results indicate that intrathecal Rh iduronidase injections are an effective way of administering protein across the blood-brain barrier. The protein is detectable both on the surface of brain cells and in lysosomal brain cells, as shown in even method 2, which shows that intrathecal injection is an effective means of transporting enzymes involved in lysosomal storage disorders, can be administered spinally and provide therapeutic benefit to the subject disease.

EXAMPLE 4

Spinal injection of Rh-iduronidase improves MPS and symptoms [0130] Preliminary experiments have shown that iduronidase administered intrathecal injection has successfully crossed the blood-brain barrier can be detected in significant amounts in neurosyss lysosomes on the surface of cerebral cortex, and also penetrates deep brain tissues. Based on these results, it appears that LVD impairs brain function, which can be treated by replacing spinal injection with enzyme injection for treatment.

[0131] To assess the efficacy of spinal injection of enzymes involved in lysosomal storage disorder, canine subjects affected by MPS I storage lysosomal disorder and lacking the enzyme iduronidase where they were treated by spinal administration of Rh iduronidase. Iduronidase levels in brain and central nervous system tissues evaluated after 4 weeks of treatment.

[0132] As noted above, a dose of 1 mg rh-iduronidase / injection was selected. Four MPS-affected animals were treated with 1 mg rh-iduronidase / injection by reservoir injection once weekly for four weeks and enzyme levels measured 48 hours after the last treatment dose. Spinal injections resulted in extensive enzyme distribution in the brain, spinal cord and meninges. Detection of enzyme activity in MPS I animals revealed a mean 21-fold increase in iduronidase levels in these animals compared to the control group. Analysis of the enzyme activity in the deep brain tissue and surface brain of animals with MPS I showed an average 11-fold and 37-fold increase in activity, respectively.

[0133] In a further set of experiments, the total cerebral enzyme activity in four treated dogs reached an average of 277 Units / mg compared to an average level of 11.9 Units / mg in untreated normal dogs, and an average of 23 times normal with a range of 17-34 fold of normal levels. As with normal dogs, α-L-iduronidase activities were higher (3-4 times) on the brain surface than in its internal regions (474.0 ± 257.7 vs. 138.7 ± 93.5). Nevertheless, levels in the deep brain were still above 11 times normal.

[0134] Because intrathecal administration of the protein places the protein directly in the PMR, which washes the entire central nervous system, it is likely that any protein injected by this route is detectable in all areas of the CNS. The MPS I animals used above were used to assess the presence of iduronidase in the spinal cord and meninges of treated animals as described above in Example 1.

[0135] Samples of the spinal cord and meninges were obtained from four animals with MPS I and iduronidase activity as above (mean of cervical, thoracic and lumbar regions) was measured. Levels of iduronidase activity in the spinal cord in MPS I animals were on average 13 times higher than in control animals, while the enzyme levels were approximately 300 times higher in MPS I animal spinal tires. In repeated experiments, rh-iduronidase levels in the spinal cord in spinal-treated dogs with MPS I reached an average of 160 units / mg or about 13 times the normal level of 11.7 units / mg (p = 0.022, Table 2). The enzyme penetration was better in the cervical and thoracic regions than in the lumbar spine probably due to incomplete distribution of the enzyme from the large reservoir injection site. The rhIDU levels in treated dogs with MPS I were 17 times normal in the cervical spinal cord, 18 times in the thoracic spine, and approximately 5 times in the lumbar spine. In core tires, rh-iduronidase levels reached an average of 4.780 units / mg or were more than 300 times higher than normal levels of 15.4 units / mg (p = 0.0018, Table 2). Even in animals with the lowest level of enzyme penetration, iduronidase levels in the tires were 160 units / mg or 140 times normal levels.

Table 2: Iduronidase levels in IT treated dogs with MPS I (dose ~ 1 mg weekly)

<td>Place of the CNS</td><td>IT treated [range] (n = 4)</td><td>Untreated / treated with placebo Normal (n = 5)</td><td>The ratio of IT treated vs Normal</td>

<td>Brain</td><td> 277 ± 89,1 [203 403]</td><td> 11,9 ± 1,95</td><td>23.3 p = 0.0003 *</td>

<td>Spinal cord</td><td></td><td></td><td></td>

<td>cervical</td><td> 196 ± 133 [43,3 367,3]</td><td> 11,1 ± 1,69</td><td> 17,7</td>

<td>chest</td><td> 224 ± 138 [132,4 428,7]</td><td> 12,0 ± 3,10</td><td> 18,7</td>

<td>lumbar</td><td> 59,8 ± 85,9 [8,8 188,0]</td><td> 12,1 ± 2,90</td><td> 4,9</td>

<td>Average</td><td> 160 ± 115 [73,1 328,0]</td><td> 11,7 ± 0,57</td><td>13.7 p = 0.0216 *</td>

<td colspan="2">Spinal cord tires</td><td></td><td></td>

<td>cervical</td><td> 7030 ± 3480 [4060 11,100]</td><td> 15,6 ± 4,85</td><td> 451</td>

<td>chest</td><td> 5490 ± 4200 [1570 9970]</td><td> 14,6 ± 3,34</td><td> 376</td>

<td>lumbar</td><td> 1810 ± 2690 [95,4 5820]</td><td> 16,1 ± 9,10</td><td> 112</td>

<td>Average</td><td> 4780 ± 2220,0 [2160 -</td><td> 15,4 ± 0,76</td><td>308 p = 0.0018 *</td>

7580]

Iduronidase levels are calculated from the average values ​​for each region for each dedicated dog. Iduronidase levels are expressed in units of iduronidase per mg of protein. Mean means for each animal are ± standard deviation. The ranges for each data set are the average values ​​of iduronidase analysis in each type of tissue for each dog. * Statistically significant.

EXAMPLE 5

Spinal Iduronidase treatment reduces GAG levels in animals with MPS I [0136] An important factor in weakening a subject with lysosomal storage disorders such as MPS I is the lack of macromolecule degradation resulting in the accumulation of glycosaminoglycans in cell lysosomes. It is postulated that enzyme replacement therapy by spinal injection should enhance GAG ​​degradation and restore GAG ​​levels to those comparable to normal individuals.

[0137] To test the ability of recombinant iduronidase treatment to improve GAG ​​storage in MPS I subjects, dogs with MPS I treated as above were analyzed for cerebral lysosomal glycosaminoglycan levels. Brain levels in MPS I animals receiving rh-iduronidase were reduced to normal or below normal levels, while untreated MPS I animals showed GAG levels of about 2X such as normal subjects. GAG levels measured in the spinal meninges were 7 times as normal levels in untreated MPS I animals, but reduced by 57% to 3 times as normal levels in MPS I animals receiving intrathecal iduronidase.

[0138] GAG levels were also compared in treated animals with MPS I receiving spinal or IV treatment (or single bolus, weekly bolus, monthly, quarterly bolus, bolus administered every six months, annual bolus or alternatively, continuous bolus) with rh-iduronidase ( Figure 4). GAG levels in MPS I animals receiving iduronidase IV treatment were similar to or slightly higher than levels observed in untreated MPS I animals (approximately 10 mg / mg versus approximately 8 mg / mg, respectively). Intrathecal administration of iduronidase reduces brain GAG levels to below normal, at about 4 mg / mg protein, or 2 times less than untreated MPS I animals.

[0139] In further experiments, it was again shown that multiple increases in normal levels of rh-iduronidase activity in the brains of treated dogs with MPS I led to a significant reduction in GAG levels relative to untreated control dogs with MPS I and normal GAG levels were achieved (Table 3). The mean GAG levels in the brains of dogs with MPS I treated with spinal rh-iduronidase were 4.47 ± 0.69 mg / mg dry weight compared to 8.26 ± 1.23 mg / mg for untreated dogs with MPS I (p = 0 , 0017). The level of GAG in the brains of spinal treated dogs was not significantly different from that of untreated normal dogs (5.43 ± 1.95, n = 8, p = 0.37). GAG brain levels in spinal-treated MPS I dogs were also significantly below those in MPS I dogs treated in previous studies with IV rhIDU infusions (10.4 ± 2.14, n = 12, Table 3). Because increasing age may result in increased storage, brain GAG content was also coupled to the age of control dogs, IV treated and IT treated dogs. The coupling further confirms the normalization of total GAG for IT treated dogs compared to controls or IV treated dogs of comparable age.

[0140] Tire GAG ​​levels were analyzed in samples from the cervical, thoracic and lumbar regions (Table 3). Overall, the average level of GAG in the meninges of IT treated dogs will decrease to 57%, to 15.3 mg / mg (range 9.33 to 22.5 mg / mg) compared to the average of untreated dogs of 35.9 mg / mg. This represents a decrease from 7-fold normal in untreated dogs with MPS I to 3-fold normal in treated animals and was statistically significant (p = 0.009). Samples from cervical and thoracic tires often have better GAG clearance than distal lumbar tires. The mean total GAG levels in the spinal cord of IT-treated MPS I dogs decreased to 3.43 mg / mg compared to 5.04 mg / mg for untreated MPS I dogs, but the overall levels were relatively low, and the change was not significant statistically.

Table 4: Glycosaminoglycan levels in untreated dogs with MPS I, IT treated with MPS I, IV treated with MPS I, and untreated normal dogs

<td>Place of the CNS</td><td>Untreated MPS I [range]</td><td>treated with IT MPS I [range]</td><td>Treated IT ratio MPS I to untreated MPS I</td><td>treated with IV MPS I [range]</td><td>Treated IT ratio MPS I to treated IV MPS I</td><td>untreated normal [range]</td><td>Treated IT ratio MPS I to normal</td>

<td>Brain</td><td> 826 ± 1,23</td><td> 4,47±</td><td> 0,54</td><td> 10,4 ±</td><td> 0,43</td><td> 5,43 ±</td><td> 0,82</td>

<td></td><td> [6,91- 9,56]</td><td> 0,69</td><td>* p = 0.0017</td><td> 2,14</td><td>* p = 0.0001</td><td> 1,95</td><td>p = 0.37</td>

<td></td><td>n = 4</td><td> [3,63-5,26]</td><td></td><td> [7,42-16,6]</td><td></td><td> [2,95-8,31]</td><td></td>

<td></td><td></td><td>n = 4</td><td></td><td>n = 12</td><td></td><td>n = 8</td><td></td>

<td>Spinal cord</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>cervical</td><td> 3,52 ± 0,56</td><td> 2,99 ± 0,50</td><td></td><td> -</td><td> -</td><td> 1,84 ± 0,84</td><td></td>

<td></td><td> [3,12, 3,91]</td><td> [2,71-3,73]</td><td></td><td></td><td></td><td> [0,93 - 2,59]</td><td></td>

<td>chest</td><td> 5,50 ± 0,77</td><td> 2,56 ± 0,77</td><td></td><td> -</td><td></td><td> 2,11 ± 1,03</td><td></td>

<td></td><td> [4,95, 6,04]</td><td> [1,90 - 3,68]</td><td></td><td></td><td></td><td> [1,29 - 3,26]</td><td></td>

<td>lumbar</td><td> 6,11 ± 1,47</td><td> 4,75 ± 1,08</td><td></td><td> -</td><td></td><td> 5,49 ± 0,63</td><td></td>

<td></td><td> [5,07, 7,15]</td><td> [3,53 - 5,73]</td><td></td><td></td><td></td><td> [4,81 - 6,06]</td><td></td>

Spinal cord

<td>Average</td><td> 5,04±0,93</td><td> 3,43 ± 0,72</td><td> 0,68 -</td><td> 3,14±0,81</td><td> 1,09</td>

<td></td><td> [4,38, 5,70]</td><td> [2,72 - 4,38]</td><td>p = 0.075</td><td> [2,34 - 3,97]</td><td>p = 0.64</td>

<td></td><td>n = 2</td><td></td><td></td><td>n = 3</td><td></td>

Spinal cord tires

<td>cervical</td><td> 20,0±1,98 [18,6, 21,4]</td><td> 10,8 ± 2,45 [9,07 - 14,4]</td><td colspan="2"> - 4,51±0,51 [3,92 - 4,86]</td>

<td>chest</td><td> 40,5±0,95</td><td> 13,4 ± 4,30</td><td> -</td><td> 4,35±1,47</td>

<td></td><td> [39,8, 41,2]</td><td> [7,05 - 16,4]</td><td></td><td> [3,05 - 5,94]</td>

<td>lumbar</td><td> 47,2±12,0</td><td> 21,6 ± 13,8</td><td> -</td><td> 5,17±2,53</td>

<td></td><td> [38,6, 55,7]</td><td> [10,6 - 41,7]</td><td></td><td> [2,33 - 7,21]</td>

<td>Average</td><td> 35,9±3,03</td><td> 15,3 ± 5,56</td><td> 0,43 -</td><td> 4,68±0,97 3,26</td>

<td></td><td> [33,8, 38,0]</td><td> [9,3 - 22,5]</td><td>p = 0.009 *</td><td>^{[3,75 - 5,69]}_{p = 0.024 *}</td>

<td></td><td>n = 2</td><td>n = 4</td><td></td><td>n = 3</td>

GAG levels are calculated from the average values ​​for each region for each dedicated dog and are expressed in mg / mg dry weight. Mean means for each animal are ± standard deviation. Weekly doses of IV treatment ranged from 0.5 to 2.0 mg / kg for 3-15 months. * Statistically significant [0141] These results show that MPS I spinal treatment is an effective agent for reducing debilitating storage in these lysosomal disorders, such as accumulation of macromolecules in tissue lysosomes, and is more effective in reducing GAG levels than standard IV administration of iduronidase enzyme replacement therapy .

Example 6

Formation of lysosomal pathology after spinal rhIDU (administration) [0142] In canine MPS I, the most prominent lysosomal storage in brain histology is present in perivascular mesenchymal cells that lie close to the cerebral capillaries, separated from the bloodstream by the blood-brain barrier. To determine the extent of storage disease in affected MPS and animals, pathological analysis was performed by electron microscopy to detect GAG deposits in brain tissue. Animals with MPS I were treated with 1 mg iduronidase at weekly doses (4x) as described above in Example 1.

[0143] Tissues taken from untreated MPS I animals with perivascular macrophage disease have marked GAG storage in adventitious cells while treated animals have space around storage vessels without GAG storage (Figure 5 and Figure 6). Analysis of the pathology of neuronal disease in MPS I animals reveals that untreated animals have GAG ​​and ganglioside storage, whereas animals treated with iduronidase have dense granules with minimal storage of macromolecules (Figure 7). Ultrastructurally on electron micrographs the total amount of storage in neurons in untreated dogs with MPS I was modest. Membrane-associated, granular, flocculent, membrane, cytoplasmic and lamellar-related neuronal storage was reduced in treated dogs with MPS I. However, aggregates of electronically dense, complex, lipofuscin-like material remained in treated MPS I. animals Brain sections assessed for meningeal disease in treated or untreated MPS I animals showed the presence of large foam cells in the tires of untreated animals while the tires of iduronidase treated animals were free of devoured GAG-containing foam cells (Figure 8). Brain sections of treated MPS I animals had lymphocytic infiltration in the meninges (Figure 9). Thus, spinal-treated MPS dogs had a staggering reduction in storage in perivascular cells in both the surface and deep areas of the brain (see Figures 5 and 6). GAG storage was also reduced in brain glia of spinal-treated MPS I dogs. Focal reduction in neocortical GAG storage was also seen in three out of four MPS I dogs treated with IT. GAG storage was also reduced in the spinal meninges of animals treated with toluidine blue stained thick sections. Foam cells of the spinal cord tires were less frequently observed in four dogs with MPS I treated with rhIDU than in untreated dogs with MPS I dogs, and there was some mosaicism in the clearance pattern.

[0144] General indications are that intrathecal administration of iduronidase facilitates the clearance of glycosaminoglycans for the brain and meninges of treated subjects, reducing levels to those observed in normal subjects. it has also been observed that spinal delivery of the enzyme causes lymphocyte infiltration into the meninges, probably generating an immune response that is effective in cleansing inadequate storage of materials. Analysis of clinical signs of lysosomal storage disorders showed that spinal iduronidase treatment of MPS I animals reduced spinal compression-induced weakness and abolished nystagmus in these animals.

[0145] The effectiveness of spinal iduronidase treatment over standard IV techniques indicates that this method of enzyme replacement therapy is effective for alleviating the symptoms of MPS I subjects and is now applicable to the other common lysosomal storage disorders described above.

Example 7

Immune Response and Other Adverse Effects [0146] Moderate levels of anti-rh-iduronidase antibody were detected in both serum (up to 202 units / mL) and PMR (up to 82.0 units / mL) of two dogs with MPS I and one normal dog treated with rhIDU (Table 4). All three of these animals had prior intravenous (administered) rhIDU exposure months prior to study admission. For other treated animals, low serum levels of rhIDU antibodies were detected (3.61 to 40.9 units / mL at the end of the study), and lower levels were detected in PMR (1.39 to 2.28 units / mL). Modest increases in the number of leukocytes in PMR were observed in treated dogs. In all dogs (normal and MPS I) treated with IT rhIDU, there were different B cell accumulation, plasma cells and other lymphocytes in the spinal cord, spinal dural areas and around the brain. These dura infiltrations were typically the most intense around the roots of the spinal nerves in more severely affected cases, expanded to adjacent dural fat and connective tissue. In one of these cases, there was also moderate focal lymphocytic arteritis outside the hard dermatitis. There was no meningitis or inflammation in untreated animals, except for one normal dog that received the vehicle. Two normal dogs treated with rhIDU developed mild meningitis. The extent of the CNS inflammatory response varied among dogs and appeared to be dose dependent. No clinically apparent effects of the immune response were observed; the dogs seemed to be well and active.

Table 5: ELISA titers of rhIDU antibodies in PMR of IT-treated dogs

Dog

Week 1 Week 2 Week 3 Week 4 End

<td colspan="6">treatment</td>

<td colspan="6">Treated IT dogs with MPS</td>

<td>Ohm</td><td> 0,001</td><td> 0,000</td><td> 0,166</td><td> 2,09</td><td> 2,28</td>

<td>oz</td><td> 0,007</td><td> 0,000</td><td> 0,046</td><td> 1,08</td><td> 1,57</td>

<td>Taff</td><td> 0,000</td><td> 0,473</td><td>NW</td><td> 45,0</td><td> 52,0</td>

<td>vkf</td><td> 0,000</td><td> 4,16</td><td> 53,1</td><td> 81,5</td><td> 82,0</td>

<td>Letcho</td><td>ne IT normal dogs</td><td></td><td></td><td></td><td></td>

<td>Xu</td><td> 0,011</td><td> 0,008</td><td> 0,014</td><td> 1,35</td><td>NW</td>

<td>xi</td><td> 0,012</td><td> 0,015</td><td> 0,024</td><td> 2,49</td><td>NW</td>

<td>Bu</td><td> 0,030</td><td> 0,009</td><td> 0,670</td><td> 1,43</td><td> 1,39</td>

<td>caf</td><td> 0,105</td><td> 3,08</td><td> 31,1</td><td> 30,7</td><td> 32,8</td>

<td>dv</td><td> 0,007</td><td> 0,000</td><td> 0,526</td><td> 1,57</td><td> 1,90</td>

<td>df</td><td> 0,004</td><td> 0,000</td><td> 0,025</td><td> 0,670</td><td> 2,25</td>

<td colspan="6">Titers expressed in OD units per mL of undiluted PMR. NW = not done. * Om, Oz and Bu received 1.08 mg, Ta, Vk, and Ca received 1.38 mg, Xu and Xi received 4.14 mg, and Dv and Df received 0.46 mg IT rhIDU. fTa, Vk, and Ca were exposed to rhIDU months before the study</td>

<td> [0147]</td><td colspan="2">Giving any</td><td colspan="3">protein product carries a risk of response</td>

immune, or in the form of chronic antibody production or inflammatory response. As shown above, immune responses were observed in dogs treated with spinal iduronidase rh. Antibodies to α-L-iduronidase were detected in the serum and PMR of three dogs that had exposure to intravenous (administered) enzyme prior to this study. Apart from lymphoplasma infiltration, there were no obvious clinical adverse reactions of the immune response.

[0148] Based on spinal treatments of MPS and other disorders as described herein can be advantageously administered in combination with a regimen that produces immune tolerance to the delivered substance. Particularly contemplated immune tolerance methods include, e.g., those described in US Patent Publication 20030211113 and US Patent Publication 20040009906, each incorporated herein in its entirety by reference. Downstream representatives of immune tolerance protocols are provided in Example 9.

Example 8

Treatment of MPS Subjects and Recombinant Iduronidase [0149] Effective treatment of MPS I dogs with recombinant human iduronidase indicates that spinal enzyme replacement therapy provides effective treatment for human subjects with MPS I.

[0150] For the treatment of patients with MPS and rh-iduronidase, patients with mucopolysaccharidosis I are selected. The subjects are evaluated at the beginning and at 6, 10, 14, 18, 22, 26, and at least once a month for 52 weeks for detailed clinical tests, magnetic resonance imaging of the abdomen and brain, echocardiography, range of motion measurements, polysomnography, clinical laboratory assessments, measurements of leukocyte activity of α-L-iduronidase and urinary secretion of glycosaminoglycan. Subjects should also be evaluated for CNS symptoms that result from lysosomal storage of brain granules. Such symptoms include developmental retardation and / or regression in the development of a diseased subject that can be clinically assessed, for example, using the Bayley Infant Development II Scale (including motor and developmental quotient monitoring), language monitoring or other intellectual and motor development features , monitoring of evoked potential tests, e.g. auditory or other evoked potential testing. Another symptom, high pressure hydrocephalus caused by the presence of storage granules in the meninges near the arachnoid granules, can be clinically monitored and evaluated by methods known in the art for determining PMR pressure by lumbar puncture and / or by an intraventricular catheter. Lysosomal storage in the cervical meninges near the C1-C5 spinal cord or elsewhere in the spinal cord can also be clinically evaluated, which manifests as progressive compression spinal cord compression with lower extremity weakness, loss of bladder and bowel control, and sensory deficits can also be monitored . Such symptoms can be monitored by using e.g. neurological examination of Babinski's abnormal reflexes, deep tendon reflexes, motor function or sensation. Neurophysiological deficits in spinal cord compression can be assessed using somatosensory evoked potentials. Magnetic resonance imaging with or without contrast agent can also be used to identify the anatomical location of the compression, as well as assess swelling or other indications of spinal injury at the site of compression. Perivascular storage of lysosomal storage granules can be assessed by determining the presence of cysts around the vessels, which can also be assessed using MRI scans to determine the size and number of such cysts. Monitoring these symptoms before and after treatment allows you to evaluate the effectiveness of therapeutic intervention.

[0151] Iduronidase was administered to subjects by intrathecal infusion (diluted in physiological saline 0.1 percent human serum albumin) at a dose of, for example, from 1 mg of iduronidase per 20 kg of animal weight, delivered once weekly. Intrathecal administration is done by direct injection into PMR or as in Penn et al. (Neurosurgery. 40.94-9. 1997), with a drug pump implanted into the lumbar subarachnoid space, e.g. Medtronic SYNCHROMED® pump or similar device for intrathecal delivery. The pump is implanted according to the manufacturer's instructions and can be implanted at any level appropriate to the individual or disorder being treated. For example, the pump catheter tip can be located on the T-10 vertebra of the spine. Subjects are premedicated with diphenhydramine (0.5 to 1.25 mg per kilogram body weight).

[0152] In initial therapy, iduronidase is administered to affected subjects once a week for four weeks. Administration may be continued for a longer period of time, depending on the severity of the MPS I disease in the subject being treated, as well as the subject's age, weight and sex. Dosage amounts and duration can be determined by the attending physician.

[0153] Subjects are assessed for changes in MPS I symptoms using motor skills tests, MRI analysis of tissue GAG ​​sediments, and urinary GAG levels at the time points mentioned above. For example, urinary GAG levels in patients with MPS-I are compared to normal secretion values. There is a wide range of GAG values ​​in the urine of untreated subjects with MPS-I. A reduction of more than 50% in the secretion of non-decomposed GAGs after rh-iduronidase therapy is the obligatory way to measure a subject's response to treatment. For example, data is collected in measuring leukocyte iduronidase activity and oral iduronidase activity before and after therapy in subjects with MPS I.

[0154] Increased motor ability or reduction of GAG deposits in the brain or GAG urine levels indicate that treatment with rh-iduronidase breaks excess GAG in treated subjects and alleviates disease symptoms.

EXAMPLE 9

Specific antigenic tolerance and enzyme replacement therapy in the treatment of lysosomal storage disorders.

[0155] As noted above, intrathecal iduronidase treatment of MPS-affected animals

And caused lymphocytic penetration into the meninges of treated animals. This may be due to the immune system overreacting to the presence of large amounts of foreign antigen delivered to the animal. To overcome this type of reaction, specific antigenic tolerance methods were used to effectively suppress the immune system. Another US patent application presents a treatment regimen for dogs with MPS I, which means induction of antigen-specific tolerance and intravenous replacement therapy with iduronidase. Based on the results described herein, which indicate that intrathecal administration of the enzyme is more effective than intravenous injection in reducing brain storage of GAG and reducing the clinical symptoms of MPS I, it follows that the use of intrathecal injection in combination with specific antigen tolerance will provide greater relief to sufferers on MPS L.

[0156] Mucopolysaccharidosis subjects are selected for treatment. Subjects are evaluated at the beginning and after 6, 12, 26 and 52 weeks according to detailed clinical tests, magnetic resonance imaging of the abdomen and brain, echocardiography, range of motion measurements, polysomnography, clinical laboratory tests, measurements of leukocyte α-liduronidase activity and glycosaminoglycan secretion.

[0157] Cyclosporin A (Neoral or Sandimmune) and Azathioprine (Imuran) are obtained from commercial sources. Both drugs are administered orally at a dose and frequency as follows: CsA Neoral® 12.5 mg / kg / daily, divided by each bid; Aza Imuran® 5 mg / kg qod for two weeks stabilization. Drugs are then given at this dose for an additional two weeks in the presence of toleragen. The doses of all drugs are halved every 2 weeks after the first toleragen infusion. Entities are monitored for adverse reactions and at peak / maximum and minimum CsA levels. CsA is preferably higher than 400ng / ml.

[0158] Recombinant α-L-iduronidase is produced in Chinese hamster ovary cells using bioreactors and standard column chromatography, and extensively analyzed for safety and purity. Α-L-iduronidase activity is measured according to the method of Shull et al. supra., or by analysis, the results of which are presented in SI units (Kakkis et al. Mol Genet Metab. 2001, 72 (3): 199-208; Kakkis et al., N Engl J Med. 2001; 344 (3) : 182-8). When the second analysis is used, the dose of 125,000 U α-L-iduronidase per kilogram corresponds to 100 SI units per kilogram. Urinary glycosaminoglycan excretion is measured in accordance with the adaptation of the Bjornsson method. Analyzes of the immunosorbents attaching enzymes for antibodies to α-Liduronidase use a variation of the method of Shull et al., And Western blotting is performed by a standard method.

[0159] Toleragen is administered by intravenous infusion (diluted in physiological saline with 0.1 percent human serum albumin) at a dose of 0.056 mg / kg, delivered once weekly. After adaptation to iduronidase, intrathecal treatment with the enzyme composition is as described herein. In a preferred embodiment, the methods of this example have been tested in dogs treated monthly, using 1 mg of rh iduronidase injection as toleragen. After 4 injections over a three-month period, GAG levels in these animals' brains were observed as normal. The administration protocol is preferably effective such that 1 mg injections are given quarterly or every 6 months.

[0160] Intrathecal administration for use in the methods of the invention is by direct injection into PMR or as in Penn et al. (Neurosurgery. 40: 94-9. 1997), with a drug pump implanted into the lumbar subarachnoid space, e.g.

Medtronic SYNCHROMED pump^® or similar device for intrathecal delivery.

The pump is implanted according to the manufacturer's instructions and can be implanted at any level appropriate to the individual or disorder being treated. For example, the pump catheter tip can be located on the T-10 vertebra of the spine. The first dose is given at the end of the two-week stabilization period, and then weekly. Subjects are premedicated with diphenhydramine (0.5 to 1.25 mg per kilogram body weight).

[0161] After induction of tolerance, preferably about 12 weeks after the start of the stabilization period, the dose is increased to one per week, 0.58 mg per kilogram.

[0162] Subjects are evaluated for changes in one or more indicators of brain disease symptoms associated with lysosomal storage disease after treatment with recombinant iduronidase. Such indicators include, but are not limited to changes in development, motor function, maintaining development over time, lowering CSF pressure, reducing neurological symptoms through ailments or by examination, reduced spinal pressure in the assessment of MRI of the neck or spine, and somatosensory evoked potentials after treatment recombinant iduronidase. It is anticipated that iduronidase treatment increases the distribution of excess GAG in the brain and spinal cord of affected individuals and reduces the pressure exerted on the spinal cord. The improvement in motor skills is an indicator of a reduction in spinal compression as a result of iduronidase treatment.

EXAMPLE 10

Intrathecal treatment of other lysosomal storage diseases.

[0163] The above methods are useful in the treatment of human subjects manifesting the clinical phenotype of deficiency of any lysosomal enzyme. All subjects show some clinical evidence of the accumulation of visceral and soft tissue glycosaminoglycans or other macromolecules with varying degrees of functional impairment. Diseases that are treated or prevented using the methods of the invention are: mucopolysaccharidosis II (MPS II), MPS IIIA, MPS IIIB, metachromatic leukodystrophy (MLD), Krabbe, Pompe, Ceroid Lipofuscinosis, Tay-Sachs, Niemann-Pick A and B Gaucher disease and other lysosomal diseases as described above.

[0164] For each disease, the enzyme administered in intrathecal enzyme replacement therapy or stabilization regimen will contain a specific compound or enzyme. For methods involving MPS II, the preferred compound or enzyme is iduronato-2sulfatase. For methods involving MPS IIIA, the preferred compound or enzyme is heparan-N-sulfatase. For methods involving MPS IIIB, the preferred compound or enzyme is α-N-acetylglucosaminidase. For methods involving metachromatic leukodystrophy (MLD), the preferred compound or enzyme is arylsulfatase A. For methods involving Krabbe, the preferred compound or enzyme is galactosylceramidase. For Pompe methods, the preferred compound or enzyme is acid α-glucosidase. For CLN-related methods, the preferred compound or enzyme is tripeptidyl peptidase. For methods involving TaySachs, the preferred compound or enzyme is hexosaminidase alpha. For Niemann-Pick A and B methods, the preferred compound or enzyme is acid sphingomyelinase.

[0165] The enzyme may be administered in dosages suitable for the subjects to be treated and generally delivered on a mg / kg basis as described in the Detailed Description. Subjects receiving the enzyme are monitored for levels of enzymes in the blood and tissue samples and other symptoms, especially the lysosomal storage disorder being treated. For example, subjects with Gaucher disease (type 3) that exhibit reduced motor skills or myoclonic seizures due to abnormal lipid storage are monitored for improved motor skills and reduced seizure / seizure frequency after intrathecal glucoerebrosidase replacement therapy.

[0166] The improvement in one or more symptoms of lysosomal storage disorder after spinal administration of an enzyme deficient in lysosomal storage disorder shows that this route of administration is a new and useful way to treat lysosomal disorders affecting human subjects.

EXAMPLE 11

Monthly intrathecal regimen [0167] As discussed herein, intrathecal administration of rhIDU has demonstrated effective CNS penetration. In some exemplary studies, CNS penetration and a reduction in glycosaminoglycan storage (GAG) in mucopolysaccharidosis I (MPS I) in dogs have been demonstrated following intrathecal administration of weekly doses of approximately 1 mg of rhIDU. Further studies described in this example show that monthly rather than weekly treatments are also effective.

[0168] Three dogs with MPS I received 4 monthly doses of ~ 1 mg IT rhIDU in combination with weekly IV rhIDU. In this combined regimen, it was observed that iduronidase levels exceeded 23-fold normal levels in the brain, 7-fold in the spinal cord and 423-fold levels in the meninges of treated dogs monthly compared to 23-fold, 13-fold and 300-fold respectively times in 4 dogs treated weekly only with intrathecal rhIDU. GAGs in the brain have reached normal levels in both regimens. With monthly treatment, 51% reduction in brain GAG storage (46% observed for weekly IT administration for comparison; Figure 10A), 22% reduction in spinal cord GAG (32% observed for weekly IT administration for comparison; Figure 10B) and 22 The% reduction in meningeal GAG (for comparison, 57% with IT weekly administration; Figure 10C) was compared with 4 untreated dogs with MPS I. There were no significant differences in iduronidase levels or GAG levels at monthly compared to weekly IT rhIDU. Therefore, monthly intrathecal administration may be used.

[0169] Animals were examined for inflammatory responses and for inducing tolerance. One dog developed meningitis in the meninges and a mild antibody response in the blood and PMR. One dog had neurological symptoms (see table below) at the start of treatment, but these symptoms improved after 4 monthly IT IT rhIDU doses with simultaneous weekly IV rhIDU. _

<td>IN FRONT OF</td><td>AFTER</td>

<td>Lethargic</td><td>Alert</td>

<td>Atactic gait</td><td>No ataxia</td>

<td>Gag reflex absent</td><td>Vomiting reflex present</td>

<td>Tilting the head</td><td>Without tilting your head</td>

[0170] A second dog was made tolerant to rhIDU using a new method (described e.g. in joint application US 10 / 141.668 filed on May 6, 2002 and 10/429314 filed on May 5, 2003 (published as US Patent Publication US 20030211113 and U.S. Patent Publication 20040009906, each incorporated herein in its entirety by reference), and it had little or no detectable immune response in the blood and in PMR and very mild meningitis. Treated dogs had histologically reduced GAG storage in leptomeningeal and perivascular. The fact that GAG storage is visibly reduced in perivascular cells, glia, and leptomeninges neocortex in treated IT dogs is depicted in Figure 11A (untreated has swollen, foamed, GAG loaded cells) and 11B (treated; thin cells with significantly less GAG storage ). Including tolerance induction, the data illustrated in Figures 12A-12D show that an animal that was premedicated with an immunosuppressive scheme and became tolerant to rhIDU has a much milder immune response to rhIDU therapy. Figures 12A and 12C show that animals treated with rhIDU alone develop lymphocytic and plasmocytal infiltration (Figures 12A and 12C). On the other hand, premedication with a scheme for inducing immune tolerance greatly reduces this response (Figure 12B and Figure 12D).

[0171] These studies show that monthly IT rhIDU can be as effective as weekly IT rhIDU in correcting lysosomal storage in the brain and meninges of dogs with MPS.

[0172] The above illustrates and illustrates the invention, but is not intended to limit the invention as defined by the following claims.

Prepared and verified

Mirosława Ważyńska

Patent Attorney