KR20130136990A - Use of lysosomal acid lipase for treating lysosomal acid lipase deficiency in patients - Google Patents

Abstract

The present invention provides a method for treating LAL deficiency comprising administering to a mammal a therapeutically effective amount of lysosomal acid lipase at an effective dosage frequency. Methods are provided for improving growth and liver function, increasing LAL tissue concentration, and increasing LAL activity in human patients with LAL deficiency.

Description

USE OF LYSOSOMAL ACID LIPASE FOR TREATING LYSOSOMAL ACID LIPASE DEFICIENCY IN PATIENTS}

Lysosomal Acid Lipase (LAL) deficiency is a rare lysosome characterized by a failure of cholesteryl esters (CE) and triglyceride (TAG) degradation in lysosomes due to an enzyme deficiency. Lysosomal storage disease (LSD). LAL deficiency is similar to other lysosomal storage disorders with accumulation of matrix in many tissues and cell types. Substrate accumulation in LAL deficiency is most pronounced in cells of the reticuloendothelial system, including hepatocytes in the liver Kupffer cells, spleen and small intestine lamina propria. Reticulum endothelial cells are known as macrophage mannose / N-acetyl glucosamine receptors (also known as macrophage mannose receptors, MMR or other) that mediate binding, cell uptake and lysosomal internalization with GlcNAc or mannose terminated N-glycans. Known as CD206), providing a pathway for potential modification of enzyme deficiencies in these important cell types.

LAL deficiency is a multi-line disease most commonly caused by gastrointestinal, liver and cardiovascular complications and is associated with significant morbidity and mortality. The clinical effect of LAL deficiency is due to a tremendous impairment in cholesterol and lipid homeostasis mechanisms, including the huge accumulation of lipid material in lysosomes in many tissues and a substantial increase in cholesterol synthesis in the liver. LAL deficiency exists as at least two phenotypes: Wolman Disease (WD) and Cholesteryl Ester Storage Disease (CESD).

The moon disease is named after the doctor who first described it and is the most aggressive being of LAL deficiency. This phenotype is characterized by gastrointestinal and liver signs, including growth insufficiency, malabsorption, fatty malaise, immense weight loss, lymphadenitis, splenomegaly and hepatomegaly. Moon moon disease is fast and usually dies within one year of life. Case report reviews indicate that survival within 12 months is extremely rare for patients with growth failure due to severe LAL deficiency. In this most aggressive form, growth failure is a predominant clinical feature and an important contributor to premature death. Liver involvement, evidenced by liver enlargement and elevation of transaminase, is also common in infants.

Diagnosis of moonshine is established through both physiological findings and laboratory analysis. Infants typically are hospitalized within the first two months of life because of diarrhea, persistent vomiting, difficulty eating, dwarf growth and cessation of growth. Physical findings include abnormal dilatation by hepatomegaly and splenomegaly, and radiographic examination often indicates calcification of the adrenal glands. Laboratory evaluations typically show elevated levels of serum transaminase and no or significantly reduced endogenous LAL enzyme activity. Elevated blood levels of cholesterol and triglycerides are seen in some patients.

Patients with LAL deficiency may also be present in later life with predominant liver and cardiovascular involvement, often referred to as cholesteryl ester reservoir (CESD). In CESD, the liver is severely affected by marked hepatomegaly, hepatocellular necrosis, elevation of transaminase, cirrhosis of the liver and liver fibrosis. Due to increased levels of CE and TAG, cardiovascular involvement may be characterized by hyperlipidemia. Accumulation of fat deposits on arterial walls (atherosclerosis) has been reported in some subjects with CESD. Sediments narrow the arterial lumen and can cause vascular occlusion that increases the risk of significant cardiovascular events, including myocardial infarction and stroke. However, not all subjects with LAL deficiency develop atherosclerosis. For example, patients with moon sickness are plagued by other symptoms associated with the disease, including enlarged liver and spleen, lymphadenitis, and poor absorption by the small intestine, but WD is generally not characterized by atherosclerosis ( The Metabolic and Molecular Bases of Inherited Disease (Scriver, CR, Beaudet, AL, Sly, WS and Valle D., eds) 7th ed., Volume 2 p. 2570 McGraw-Hill, 1995). Likewise, not all CESD patients show atherosclerosis. See Bi Biseglie et al., Hepatology 11 : 764-772 (1990), Ameis et al., J. Lipid. Res . 36: 241-250 (1995). The pattern of CESD is highly variable because some patients are not diagnosed until later complications, while others may develop liver dysfunction in infancy. CESD is associated with shortened lifespan and significant poor health. The life expectancy of patients with CESD depends on the severity of the complications involved.

Current treatment options for moonshine are very limited. Antibiotics are given to infants with evidence of fever and / or infection. Steroid replacement therapy and ad hoc nutritional support for adrenal insufficiency may be prescribed, but there is no evidence that such intervention prevents death, and it is now unclear whether they affect short-term survival. In a series of four patients with bone marrow transplant treated LAL deficiency, all four patients died due to course complications within a few months of transplantation. Although some success has been described in subsequent case reports, mortality is still high, and many patients are too poor to survive up to the pretransplant conditioning regimen and are not transplanted. Very few reported long-term survivors indicate that the modification of enzyme deficiency in hematopoietic stem cells alone is sufficient to substantially improve the clinical condition of the disease. Clinical support is typically provided through dietary restrictions in an attempt to limit the increase in untransportable and uncatalytic lipids associated with the acute symptoms of the disease causing death.

Current treatment options for the CESD phenotype include the control of lipid accumulation through diet, excluding foods rich in cholesterol and triglycerides, the inhibition of cholesterol synthesis and the cholesterol-lowering drugs (eg, statins and cholestyramine). Emphasis is placed on symptomatic therapy through the production of apolipoprotein B through administration. Although some clinical improvement may be seen, the symptoms of the disease being experienced persist and disease progression still occurs.

It has been suggested that enzyme replacement therapy with recombinant LAL may be a viable treatment option for lysosomal acid lipase deficiency and related diseases (Meyers et al. (1985) Nutrition Res . 5 (4): 423-442; WO9811206; And Besley (1984) Clinical Genetics 26: 195-203). Some studies using a mouse model of LAL deficiency have demonstrated some abnormal fertilization of LAL deficient (LAL ^-/- ) mice via high doses of recombinant human LAL (> 1 mg / kg body weight) once every three days. (See, eg, US Pat. No. 2007/0264249 to Grabowski). This early study to correct defects in LAL deficient mice suggests that relatively high amounts and frequent dosages of recombinant LAL protein are required to correct the phenotypes being experienced. Unlike the LAL ^-/- rat model first described by Yoshida and Kuriyama (1990) Laboratory Animal Science, vol 40, p 486-489, the LAL ^-/- mouse model used in this study is strictly related to human WD. It is important to note that dissimilarities, ie, LAL deficient mice, do not exhibit growth defects seen in human patients.

To date, exogenous LAL has not been administered to humans and no effective treatment is available for the treatment of LAL deficiency, including WD, CESD and others. Thus, there is a tremendous need for treatment with minimal dosing frequency in order to improve the quality of life of patients. In addition, therapeutically effective doses that restore growth, normalize liver function, increase LAL tissue concentration, and increase LAL activity in human patients are desirable.

The present invention is based on the first human clinical case in which exogenous LAL was successfully administered to a patient. Infants with other lethal forms of LAL deficiency (monthly or premature LAL deficiency) were effectively treated by administering exogenous LAL, and the safety assessment of LAL enzyme replacement therapy was evaluated in a group of human patients with late LAL deficiency. Infants with premature LAL deficiency were administered weekly at low doses without causing any side effects or reactions. Clinical / experimental measurements for dramatic improvement and efficacy of vital signs were observed as early as 1-2 weeks after initial administration. After weekly dosing over 4 months, the treated infants recovered normal growth and showed significant improvement in all symptoms associated with LAL deficiency, including malabsorption, hepatomegaly and hepatic function. Later adult patients also received a small amount of exogenous LAL weekly and there were no signs of side effects. Thus, the clinical data collected so far indicates that enzyme replacement therapy using the exogenous LAL of the present invention provides a safe and effective treatment for LAL deficiency.

Accordingly, the present invention provides a method of treating a disease or condition associated with LAL deficiency in human patients by administering an effective amount of exogenous lysosomal acid lipase (LAL). The exogenous LAL can be a recombinant human LAL having an N-linked glycan structure comprising at least one mannose and / or mannose-6-phosphate. Exogenous LALs are effectively internalized, for example, into lysosomes of lymphocytes, macrophages and / or fibroblasts.

In some embodiments, the human patient with LAL deficiency is diagnosed with moon disease (WD). In one embodiment, the administration is sufficient to increase the growth of WD patients. In one embodiment, the administration is sufficient to restore normal growth of the WD patient. In another embodiment, a human patient with LAL deficiency is diagnosed with cholesteryl ester storage disease (CESD). The treatment method according to the invention can be provided to human patients of any age.

Also provided herein are methods for treating a human patient suffering from LAL deficiency by administering a recombinant human LAL to the patient in an effective amount to improve liver function. In some embodiments, the administration is sufficient to normalize the liver test. In one embodiment, the administration is sufficient to reduce serum levels of hepatic transaminase. For example, hepatic transaminase may include serum aspartate transaminase (AST) and / or alanine transaminase (ALT). In one embodiment, the administration is sufficient to minimize hepatomegaly. In one embodiment, the administration is sufficient to reduce the liver size of the patient. In one embodiment, the administration is sufficient to reduce serum ferritin levels.

In one embodiment, the administration is sufficient to reduce serum lipid levels, including, for example, cholesteryl ester (CE) and / or triglyceride (TG) levels.

Also provided are methods of increasing LAL activity in human patients with LAL deficiency. Such methods include administering a recombinant human LAL to the patient, such that the administration results in increased LAL activity if, for example, it can be measured in lymphocytes and / or fibroblasts.

In one embodiment, a method is described for treating a disease associated with LAL deficiency in a human patient by administering an effective amount of exogenous LAL protein to the patient once every 5 days to once every 30 days.

In some embodiments, a human patient with LAL deficiency is administered from about 0.1 mg to about 50 mg of exogenous LAL per kilogram of body weight. In one embodiment, the human patient is administered from about 0.1 mg to about 10 mg of exogenous LAL per kilogram of body weight. In one embodiment, the human patient is administered from about 0.1 mg to about 5 mg of exogenous LAL per kilogram of body weight.

In one embodiment, the infusion rate is about 0.1 mg / kg / hour to about 4 mg / kg / hour.

In some embodiments, the human patient is treated with a second therapeutic agent. The second therapeutic agent may include, for example, cholesterol-lowering drugs (eg statins or ezetimibe), antihistamines (eg diphenhydramine) or immunosuppressive agents.

1A shows a weekly dose of exogenous LAL (SBC-102) (dose: 0.2 mg / kg (initial infusion; week 0); 0.3 mg / kg (week 1); 0.5 mg / kg (week 2); and FIG. 3 shows levels of serum asphaltene transaminase (AST) in infant male menopausal (ie, premature LAL deficiency) patients who received 1.0 mg / kg (weeks 3-8). 1B depicts the levels of serum alanine transaminase (ALT) in the same patient. Patients are 4 months to 1 week of age at initial infusion.
Exogenous LAL (SBC-102) (dose: 0.2 mg / kg (initial infusion; week 0); 0.3 mg / kg (week 1); 0.5 mg / kg (week 2); and 1.0 mg / FIG. 3 shows the serum ferritin levels in a monthly illness with a weekly dose of kg (Weeks 3-8). Patients are 4 months to 1 week of age at initial infusion. Serum ferritin levels are shown at week 0 after the initial dose at week 0.
3 shows exogenous LAL (SBC-102) (dose: 0.2 mg / kg (initial infusion; week 0); 0.3 mg / kg (week 1); 0.5 mg / kg (week 2); and 1.0 mg / A diagram showing the growth rate of moon balm patients who received weekly doses of kg (weeks 3-8).
FIG. 4 is a diagram depicting the growth curve of a monthly pandemic (kg, percentile weight for boys).
FIG. 5 shows serum AST levels of 41 year old Caucasian male CESD patients weekly receiving a dose of 0.35 mg / kg of exogenous LAL.
FIG. 6 shows serum ALT levels in 41 year old Caucasian male CESD patients weekly receiving a dose of 0.35 mg / kg of exogenous LAL.
FIG. 7 shows serum albumin levels in 41-year-old white male CESD patients who received weekly doses of 0.35 mg / kg of exogenous LAL.
FIG. 8 shows serum ferritin levels in 41-year-old white male CESD patients who received weekly doses of 0.35 mg / kg of exogenous LAL.
FIG. 9 shows the weight gain rate in four age-matched male rats each assigned to one of four exogenous LAL dosing regimens: 1 mg per kilogram once a week, 5 per kilogram once a week. Mg, 5 mg / kg placebo once every two weeks. The numbers inside the columns represent the days after birth.
10 shows pathological and histopathological results of wild-type controls, LAL deficient rats treated with exogenous LAL and LAL deficient placebo-treated rats. Gross pathology demonstrates normalization of color and size of livestock in rats treated with exogenous LAL. Histopathology of liver tissues from rats treated with exogenous LALs shows hepatic histology that is essentially normal in contrast to the substantial accumulation of foam macrophages in placebo-treated animals.
FIG. 11 shows co-localization of recombinant human LAL (SBC-102) and lysosomal markers in lysosomes of cells examined by confocal fluorescence microscopy using a sequential scanning scheme.
FIG. 12 depicts the binding specificity of recombinant human LAL (SBC-102) to GlcNAc / mannose receptors as assessed by competitive binding assay using NR8383, a macrophage cell line.
FIG. 13 shows the activity of recombinant human LAL in normal cells and in vitro LAL-deficient cells. FIG.
14 shows the effect of recombinant human LAL (SBC-102) treatment on the internal organ mass of LAL deficient rats. Organ size is expressed as percentage of body weight determined at 8 weeks of age in LAL ^{− / −} rats and LAL ^{+ / +} rats after weekly administration of vehicle or SBC-102 at 5 mg / kg for 4 weeks.
Figure 15 is four weeks 5㎎ · kg for ¹ to a vehicle or SBC-102 after administration of each week, a view showing the body weight in wild type and LAL- deficient rats. Dose administration is highlighted on the X-axis by diamonds starting at 4 weeks.
Figure 16 is four weeks 5㎎ · kg for ¹ to a vehicle or SBC-102 after administration of each week, in WT liver cholesterol and LAL-deficient rats were determined at 8 weeks of age, cholesteryl ester and tri article illustrates the glycerides level .
FIG. 17 depicts weight gain percentages in LAL-deficient rats.
FIG. 18 shows liver weight as weight percentage in LAL-deficient rats after administration of SBC-102 for 4 weeks.
19 shows levels of tissue cholesteryl esters in LAL-deficient rats after administration of SBC-102 for 4 weeks.

The present invention provides a method of treating a human suffering from a disease or condition in response to administration of exogenous lysosomal acid lipase.

Justice

For convenience, certain terms, embodiments, and appended claims used herein are presented herein to illustrate and define the meaning and scope of various terms used to describe the present invention.

As used herein, "LAL" refers to "lysosomal acid lipase" and the two terms are used interchangeably throughout this specification. LAL may be a human protein, ie human lysosomal acid lipase. The term "SBC-102" as used herein refers to recombinant human lysosomal acid lipase. LAL is also referred to in the literature as acid cholesteryl ester hydrolases, cholesteryl esterases, lipase A, LIPA and sterol esterases.

LAL catalyzes the hydrolysis of cholesterol esters and triglycerides into free cholesterol, glycerol and free fatty acids. Thus, "LAL activity" can be measured, for example, by cleavage of the fluorescent substrate, 4-methylumbelliferyl oleate (4MUO). Cleavage of 4MUO can be detected, for example, by excitation at about 360 nm and emission at 460 nm of the emitted fluorophore, 4-methylumbelliferone (4MU). Results can be reported in relative fluorescence units (RFU). For example, the amount of substrate cleaved in a 30 minute endpoint analysis can be quantified against a 4MU standard curve, and one unit of activity (U) is defined as the amount of enzyme required to cleave 1 micromole of 4MUO per minute at 37 ° C. Can be. Thus, functional fragments or variants of LAL include fragments or variants having the ability to hydrolyze LAL activity, eg, cholesterol esters and / or triglycerides.

As used herein, “exogenous LAL” refers to LALs that are not naturally produced by a patient. For example, exogenous LAL is produced (ie expressed) in a patient as a result of administration of a recombinant LAL protein administered to the patient, an LAL protein isolated from an individual or animal and administered to the patient and LAL-encoding RNA and / or DNA. Other treatments that increase the expression of LAL protein or exogenous LAL protein.

Often referred to as medically as an IV push or bolus injection, an “intravenous injection” is a period of 15 minutes if the syringe is connected to the IV access device and the medication is direct, typically rapid and causes venous irritation or too fast effects. It refers to the route of administration sometimes injected. Once the medication is injected into the fluid flow of the IV tubing, there must be some means to ensure that the patient gets from the tubing. Usually this is done by allowing the fluid flow to flow normally, thereby delivering the medicine into the bloodstream. However, in some cases, a second fluid injection, sometimes called "flush", is used after the first injection to facilitate the influx of medication into the bloodstream.

"Intravenous infusion" refers to the route of administration for which the agent is delivered over a prolonged period of time. For example, the medicament may be delivered to the patient over a time period of 1 to 8 hours. The medicament may also be delivered to the patient over a time period of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8 hours. To achieve intravenous injection, IV gravity drop or IV pumps can be used. IV injections are typically used only at certain points in time when a patient needs a medicament, and may contain additional intravenous fluids (eg, aqueous solutions that may contain sodium, chloride, glucose or any combination thereof), such as electrolytes, It does not need to restore blood sugar and water loss.

As used herein, the term “algae” includes, but is not limited to, classifications such as chickens, turkeys, ducks, geese, quails, pheasants, parrots, finch, falcons, crows and ostrichs, liquors including emu and cassowary. Refers to any species, subspecies or variety of avian algal cavity organisms. The term is called Gallus gallus ) or chicken (e.g. white leghorn, brown leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Minorca Variety known varieties of Minorca, Amrox, California Gray, as well as varieties of turkeys, pheasants, quails, ducks, ostrichs and other poultry that normally breed in commercial quantities. Also included are individual algal organisms in all developmental stages, including embryonic and fetal stages.

The term “poultry derived” or “algae derived” refers to a composition or material produced or obtained by poultry. "Poultry" refers to birds that can be maintained as livestock, including but not limited to chickens, ducks, turkeys, quails and liquors. For example, “poultry derived” may refer to chicken derived, turkey derived and / or quail.

The term “patient,” as used herein, refers to any individual who is receiving, receiving, or willing to receive medical or treatment, for example, as directed by a healthcare provider.

As used herein, “therapeutically effective amount” refers to the dose (eg, amount and / or interval) of drug required to produce the intended therapeutic response. A therapeutically effective amount refers to a dose that results in improved treatment, cure, prevention or amelioration of a disease, disorder or side effect or reduction in the incidence or progression of a disease or disorder relative to corresponding subjects not receiving this dose. The term also includes doses effective to enhance physiological action within the scope of the present invention.

The terms “treat”, “treating” and “treatment” alleviate, attenuate or ameliorate a disease or symptom, prevent additional symptoms, ameliorate or prevent the underlying cause of the symptoms, and Inhibit the occurrence of a disease or condition, eliminate the disease or condition, cause regression of the disease or condition, eliminate the disease caused by the disease or condition, or prevent and / or develop symptoms after It refers to a method of stopping the symptoms of a disease or disorder.

As used herein in conjunction with certain terms, "kg ^-1 ", "per kg", "/ kg" and "per kilogram" refer to "per kilogram of body weight" of a mammal and therefore the terms are used interchangeably. Can be.

The term "polypeptide" as used herein is intended to include not only a singular "polypeptide" but also a plurality of "polypeptides", with monomers (amino acids) linearly linked by amino bonds (also known as peptide bonds). Refers to the molecule being constructed. The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to the specific length of the product. Thus, any term used to refer to a peptide, dipeptide, tripeptide, oligopeptide, “protein”, “amino acid chain” or a chain or chains of two or more amino acids is included within the definition of “polypeptide”, The term "polypeptide" may be used instead of or interchangeably with any of these terms. The term “polypeptide” also includes, but is not limited to, polysaccharides including glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage or modification with non-naturally occurring amino acids. It is intended to refer to the modified product after expression of the peptide. Polypeptides may be derived from natural biological sources or may be produced by recombinant techniques, but are not necessarily translated from designated nucleic acid sequences. It can be made by any method including by chemical synthesis.

As used herein, the percent homology between two amino acid sequences or two nucleotide sequences is equal to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of gaps and the number of identical positions shared by the sequences, taking into account the length of each gap (ie% homology = total # × 100 of positions of the same position). It needs to be introduced for optimal alignment of the sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the E. Meyers and W. Miller ( Comput . Appl. Biosci ., 4 : 11-17 (1988)) algorithm, which is a PAM120 weight residue table, gap length of 12 A penalty and a gap penalty of 4 were used to include in the ALIGN program (version 2.0). In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ( J. Mol , Biol . 48 : 444-453 (1970)) algorithms, which are either 16 or 16, either the Blossom 62 matrix or the PAM250 matrix. In the GCG software package (available at http://www.gcg.com) using gap weights of 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 It is included within the GAP program.

A "isolated" polypeptide or fragment, variant or derivative thereof is intended to be a polypeptide that is not in its natural environment. No specific level of purification is necessary. For example, an isolated polypeptide can be removed from its natural or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are contemplated to be isolated as disclosed herein, either natural or fractionated or partially or substantially purified by any suitable technique. This is because it is a recombinant polypeptide.

Other polypeptides disclosed herein are fragments, derivatives, analogs or variants of the aforementioned polypeptides and any combination thereof. When referring to any polypeptide disclosed herein, the terms “fragment”, “variant”, “derivative” and “analogue” refer to the corresponding natural polypeptide (eg, cholesterol esters and / or triglycerides). Any polypeptide that retains at least some of the activity of LAL polypeptide fragments, variants, derivatives and analogs that retain the ability to degrade). Fragments of polypeptides include, for example, protein fragments as well as deletion fragments. Variants of the polypeptide include fragments described above and also polypeptides having altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may occur naturally or may occur non-naturally. Non-naturally occurring variants can be generated using known mutagenesis techniques. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives are polypeptides that have been modified to exhibit additional features not found on natural polypeptides. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a subject polypeptide may contain one or more residues that are chemically derivatized by reaction of functional side groups. Also included as "derivatives" are those peptides that contain naturally occurring amino acid derivatives of one or more of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted with proline; 5-hydroxylysine may be substituted with lysine; 3-methylhistidine can be substituted with histidine; Homoserine may be substituted with serine; And / or ornithine may be substituted with lysine.

The term “polynucleotide” is intended to include not only a single nucleic acid but also a plurality of nucleic acids, and refers to an isolated nucleic acid molecule or construct, eg messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides may include conventional phosphodiester bonds or non-traditional bonds (eg, amide bonds such as those found in peptide nucleic acids (PNAs)). The term “nucleic acid” refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide. A “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA that has been removed from its natural environment. For example, recombinant polynucleotides encoding LALs contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further comprise such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may be or include a regulatory element such as a promoter, ribosomal binding site or transcription terminator.

As used herein, a "password region" is a nucleic acid moiety consisting of codons translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not translated into amino acids, they may be considered to be part of the coding region, but may be considered to be part of any adjacent sequence such as a promoter, ribosomal binding site, Transcription terminators, introns, etc. are not part of the crypto domain. Two or more coding regions of the invention may be present in a single polynucleotide construct, for example in a single vector or in separate polynucleotide constructs, for example in separate (different) vectors. Furthermore, any vector may contain a single cryptographic region or may include two or more cryptographic regions. Additionally, a vector, polynucleotide or nucleic acid of the invention can encode a heterologous coding region that is fused or not fused to a nucleic acid encoding a LAL polypeptide or a fragment, variant or derivative thereof. Heterologous coding regions include, without limitation, specified components or motifs, such as secretory signal peptides or heterologous functional domains.

Various transfer control regions are known to those skilled in the art. These include, but are not limited to, vertebrate cells, such as, but not limited to, promoters and cytomegaloviruses (rapid early promoters with intron-A), simian virus 40 (early promoters), and retroviruses (such as Raus sarcoma virus) And a transcriptional control region that acts on an enhancer fragment from. Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone and rabbit β-globin as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers as well as lymphokine-induced promoters (eg, promoters induced by interferon or interleukin).

Similarly, various transcription control elements are known to those skilled in the art. These include, but are not limited to, components derived from ribosomal binding sites, electron initiation and termination codons, and picornaviruses (especially internal ribosome entry sites or IRES, also referred to as CITE sequences).

In another embodiment, the polynucleotides of the invention are RNA, for example in the form of messenger RNA (mRNA).

The polynucleotides and nucleic acid coding regions of the present invention may be associated with additional coding regions that encode secretion or signal peptides and direct secretion of the polypeptide encoded by the polynucleotides of the invention. According to the signal hypothesis, once export of the growing protein chain across the rough endoplasmic reticulum has been initiated, the protein secreted by the mammalian cell has a signal peptide or secretory leader sequence cleaved from the mature protein. One skilled in the art will have a single peptide in which a polypeptide secreted by vertebrate cells is generally fused to the N-terminus of the polypeptide, which is cleaved from a complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. Recognize that it generates. In certain embodiments, native single peptides, such as the MKMRFLGLVVCLVLWTLHSEG (SEQ ID NO: 2) signal peptide of human LAL, are used or functional derivatives of that sequence possessing the ability to direct the secretion of a polypeptide operably related thereto. do. Alternatively, heterologous signal peptides (eg, heterologous mammalian or avian signal peptides) or functional derivatives thereof can be used. For example, the wild type leader sequence may be substituted with a leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

"Vector" means a polynucleotide comprising a single strand, double strand, circular or higher order DNA or RNA. A typical vector may contain the following conjugation elements linked by manipulation at appropriate distances to allow for functional gene expression: origin of replication, promoter, enhancer, 5 'mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and polyade Nylation Sites and Selectable Marker Sequences. One or more of these components may be omitted in specific applications. The nucleic acid cassette may comprise a restriction site for insertion of the expressed nucleic acid sequence. In a functional vector, the nucleic acid cassette contains an expressed nucleic acid sequence comprising transcription initiation and termination sites. Introns may optionally be included in the construct, eg, 5 "for the coding sequence. The vector is configured such that the particular coding sequence is located in the vector with the appropriate regulatory sequence, and the positioning and orientation of the coding sequence relative to the control sequence. The coding sequence is such that the coding sequence is transcribed under “control” of the control or regulatory sequence. It may be desirable for the modification of the sequence encoding the particular protein of interest to be made at its end, eg in some cases by modifying the sequence It may be necessary to be able to attach to the control sequence with the proper orientation or to maintain the reading frame .. Control sequences and other control sequences may be ligated to the coding sequence prior to insertion into the vector. Reading prep with and under regulatory control of the control sequences and in expression vectors already containing restriction sequences It can be cloned directly into an appropriate restriction site in the.

The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, polypeptide. The process includes, without limitation, any indication of the functional presence of the gene in the cell, including both gene knockdown as well as transient and stable expression. This includes, without limitation, transcription of genes into messenger RNA (mRNA) and translation of such mRNA into polypeptide (s). Expression of the gene produces a "gene product". As used herein, a gene product may be a polypeptide translated from a nucleic acid, eg, messenger RNA or transcript produced by transcription of a gene. The gene products described herein further bind to nucleic acids with post-transcriptional modifications such as polyadenylation or polypeptides with post-translational modifications such as methylation, glycosylation, addition of lipids, other protein subunits. , Protein cleavage, and the like.

As used herein, “host cell” refers to a cell that utilizes recombinant DNA techniques and harbors a constructed vector encoding at least one heterologous gene.

As used herein, the terms "N-glycan", "oligosaccharide", "oligosaccharide structure", glycosylation pattern "," glycosylation profile "and" glycosylation structure "have essentially the same meaning, respectively. Refers to one or more structures formed from sugar residues and attached to glycosylated proteins.

The term "pharmaceutical composition" as used herein refers to a mixture of chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients with the compounds described herein.

Insufficient LAL  Patients with activity

Without being limited to the treatment of any particular disease or group of diseases, the present invention includes the treatment of lysosomal acid lipase (LAL) deficiency in a patient. As used herein, a patient with LAL deficiency is any patient with insufficient LAL activity. Insufficient LAL activity in a patient may be low protein level or low protein activity, for example, resulting in low RNA levels. Insufficient LAL activity can result from mutations in the LAL coding sequence, the LAL regulatory sequence or in other genes (eg, genes that regulate LAL). Insufficient LAL activity can also be a result of environmental factors.

The invention can be used to treat a number of diseases in a subject or patient. Accordingly, any disease that can be advantageously treated by exogenous LAL according to the present invention is included within the scope of the present invention.

One embodiment of the present invention focuses on the treatment of lysosomal acid lipase deficiency, specifically lysosomal storage disease (LSD) resulting from moonshine disease (WD) and cholesteryl ester storage disease (CESD). Without limiting the present invention to any particular theory or mechanism of action, both WD and CESD can be attributed to mutations at the LAL locus, and the enormous accumulation of lipid material in lysosomes in many tissues and the exogenous LAL It can lead to tremendous disturbances of cholesterol and lipid homeostasis mechanisms that can be treated by administration. Thus, in one embodiment, the LAL deficiency treated according to the invention is WD. In another embodiment, the LAL deficiency treated according to the invention is CESD. In some embodiments, the diagnosis of WD or CESD is based on genetic analysis (eg, identification of functional mutations in LAL-coding sequences). In other embodiments, the diagnosis of WD or CESD is based on clinical findings (eg, physical and / or laboratory tests).

In some embodiments, exogenous LAL can be used to treat complications in a variety of diseases, such as Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASS). NAFLD refers to liver disease with similar histopathology to liver disease due to excessive alcohol intake. It is characterized by alveolar fatty liver, which causes enlargement of the liver. NAFLD can progress to NASH, which refers to a liver disease similar to NAFLD that adds inflammation and damage to the liver that can cause fibrosis and cirrhosis.

In some embodiments, exogenous LAL can be used to treat pancreatitis, such as chronic pancreatitis and / or acute pancreatitis, as well as diseases including alcohol induced pancreatic damage, such as alcohol induced pancreatitis.

Exogenous LALs produced by any useful method can be used to treat diseases caused by alcohol induced cells that result in the accumulation of lipidic esters in body tissues such as, but not limited to, liver, spleen, viscera and cardiovascular tissues Can be used. In accordance with the present invention, poor absorption can be treated by administering exogenous LAL. Exogenous LAL is useful for the treatment of patients with Tangier disease and familial hypoalphalipoproteinemia. Tangier disease / family hypophaloproteinemia is associated with the accumulation of cholesterol esters in macrophages accompanied by hepatomegaly and / or lymphadenitis with low HDL levels that can be treated by administration of exogenous LAL. For example, without limiting the present invention to any particular theory or mechanism of action, impaired LAL activity may decrease ABCA1 expression and, conversely, to patients with Tangier disease / familial hypoalphalipoproteinemia, administration of exogenous LAL The increased LAL activity obtained by increasing ABCA1 expression overcomes the effects of the ABCA1 gene with reduced functional activity as a result of polymorphism.

In some embodiments, the LAL activity level in a patient prior to treatment is about 1%, about 2%, about 3%, about 5%, about 10%, about 15%, about 20%, about 30% of normal LAL activity. , About 40%, about 50%, about 60%, about 70% or about 80%. In one embodiment, the LAL activity level in a patient before treatment is about 50% or less of normal levels of LAL activity. In one embodiment, the LAL activity level in a patient before treatment is about 40% or less of normal levels of LAL activity. In some embodiments, the LAL activity level in a patient prior to treatment is about 30% or less of normal levels of LAL activity. The LAL activity level in patients before treatment is about 30% or less of normal levels of LAL activity. In some embodiments, the LAL activity level in a patient prior to treatment is about 20% or less of normal levels of LAL activity. In some embodiments, the LAL activity level in a patient before treatment is about 10% or less of normal levels of LAL activity. In some embodiments, the LAL activity level in a patient before treatment is about 5% or less of normal levels of LAL activity. In some embodiments, the patient does not exhibit measurable LAL activity prior to treatment.

In some embodiments, LAL activity levels are measured in cultured fibroblasts obtained from human patients with LAL deficiency. In some embodiments, LAL activity levels are measured in cultured lymphocytes (eg, white blood cells) obtained from human patients with LAL deficiency. Lymphocytes include, but are not limited to, peripheral blood mononuclear cells (PMBC). Measurement methods are described, for example, in Burton et al., (1980) Clinica Chimica Acta 101 : 25-32, and Anderson et al., (1999) Mol . Genet . & Metab ., 66 : 333-345, both of which are incorporated herein by reference in their entirety. Patients with LAL deficiency treated with exogenous LAL are fibers less than about 30, about 20, about 10, about 5, about 4, about 3, about 2 or about 1 pmol / mg / min as measured using triolein as a substrate. Subcellular LAL enzyme activity. Patients with LAL deficiency treated with exogenous LALs have a leukocyte LAL of less than about 30, about 20, about 10, about 5, about 4, about 3, about 2 or about 1 pmol / mg / min as measured by triolein as a substrate. Can exhibit enzymatic activity. Patients with LAL deficiency treated with exogenous LAL can measure about 30, about 20, about 10, about 5, about 4, about 3, about 2 or about 1 pmol / mg / min as measured using cholesteryl oleate as a substrate. Less than fibroblast LAL enzyme activity. Patients with LAL deficiency treated with exogenous LAL can measure about 30, about 20, about 10, about 5, about 4, about 3, about 2 or about 1 pmol / mg / min as measured using cholesteryl oleate as a substrate. Less than white blood cell LAL enzyme activity.

Exogenous LAL Dosing

The present invention provides a method for treating a human patient with an exogenous LAL comprising administering to the patient an exogenous LAL, the administration recovering growth in the patient, improving liver function, reducing liver damage, Sufficient to increase tissue level and / or increase LAL activity. The present invention provides useful, yet uncharacterized dosing frequencies (i.e., dosing schedules) of exogenous LAL for the treatment of diseases arising from LAL deficiency, including WD and CESD, Provides uncharacterized capacity

The present invention provides a therapeutically effective amount of exogenous LAL administered to a patient once every 5 days to once every 30 days for a time period determined by one skilled in the medical sciences. In one embodiment, the time period will be the remaining life of the patient. In one embodiment, the dosing frequency is once every 5 days to once every 25 days. In one embodiment, the dosing frequency is once every 5 days to once every 21 days. In another embodiment, the dosing frequency is once every 7 days to once every 14 days. Exogenous LAL once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days , Once every 13 days or once every 14 days. In some embodiments, the exogenous LAL is administered almost weekly. In another embodiment, the exogenous LAL is administered almost every two weeks. In one embodiment, the frequency of administration is about once every 30 days.

For the treatment of a disease, the amount of exogenous LAL administered generally may vary depending on the age, health and weight of the receptor, type of concurrent treatment, frequency of treatment, and the like. Usually the dosage of active ingredient may be from about 0.01 to about 50 mg per kilogram of body weight. In one embodiment, the dosage of exogenous LAL according to the invention is about 0.1 to 0.5 mg per kilogram of body weight. In one embodiment, the dose is about 0.1 mg to about 5.0 mg per kilogram. In one embodiment, the dose is about 0.1 mg to about 5.0 mg per kilogram. In one embodiment, the dose is about 0.1, about 0.2, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45, about 0.50 mg per kilogram. In one embodiment, the dose is about 1 mg to about 5 mg per kilogram. In one embodiment, the dose is about 1 mg per kilogram. In one embodiment, the dose is about 3 mg per kilogram. For example, 0.1 mg per kilogram of body weight, 0.2 mg per kilogram of body weight, 0.3 mg per kilogram of body weight, 0.4 mg per kilogram of body weight, 0.5 mg per kilogram of body weight, 0.5 mg per kilogram of body weight, 1 mg per kilogram of body weight, per kilogram of body weight 2 mg, 3 mg per kilogram of body weight, 4 mg per kilogram of body weight or 5 mg per kilogram of body weight. In one embodiment, the dose is about 1 mg to about 20 mg body weight per kilogram.

The present invention also includes other dosages when using the dosage schedule of the present invention. For example, according to the dosing schedule of the present invention, about 0.1 mg to about 50 mg per kilogram of body weight is administered to the patient.

In some embodiments, about 0.5 to about 50 mg of exogenous LAL is administered to a diseased patient, for example only months of 1 month to 24 months of age. In one embodiment the patient is less than 1 year old. In another embodiment, the patient is less than 2 years old. In some embodiments, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 5 mg, about 10 mg, about 15 mg, about An exogenous LAL of 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg or about 45 mg is administered to a patient with moonshine. In some embodiments, about 0.5 to about 30 mg, about 0.5 to about 20 mg, about 0.5 to about 10 mg, or about 0.5 to about 5 mg is administered to a patient with moonshine. In some embodiments, about 1 to about 30 mg, about 1 to about 20 mg, about 1 to about 10 mg, or about 1 to about 5 mg.

In some embodiments, about 1 mg to about 350 mg of exogenous LAL is administered to a patient diagnosed with, for example, CESD. Thus, in some embodiments, a patient with about 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 mg exogenous LAL has CESD. Is administered. In some embodiments, about 5 to about 350 mg, about 5 to about 300 mg, about 5 to about 250 mg or about 5 to about 200 mg is administered to the patient with CESD. In some embodiments, about 10 to about 350 mg, about 10 to about 300, about 10 to about 250 or about 10 to about 200 mg are administered to the patient with CESD.

Combination therapy

Therapeutic proteins disclosed herein can be used in combination with other therapeutic agents. The present invention is provided for the pretreatment process to minimize or prevent any potential hypersensitivity reactions that may be caused by the administration of exogenous LALs according to the present invention. In one embodiment, the H-1 receptor, also known as antihistamine (eg, diphenhydramine), is administered to the patient to pretreat the potential hypersensitivity reaction. In one embodiment, the H-1 receptor antagonist is administered at a dose of about 1 mg to about 10 mg per kilogram of body weight. For example, antihistamine is administered at a dose of about 5 mg per kilogram. Administration of antihistamine may be prior to administration of the exogenous LAL according to the invention. In one embodiment, the H-1 receptor antagonist is administered from about 10 to about 90 minutes, for example from about 30 to about 60 minutes, before administration of the exogenous LAL. H-1 receptor antagonists can be administered using a migration system connected to a vascular access port. In one embodiment, the antihistamine is administered about 90 minutes prior to administration of the exogenous LAL. In one embodiment, the antihistamine is administered about 10 to about 60 minutes before administration of the exogenous LAL. In another embodiment, the antihistamine is administered about 20 to about 40 minutes before administration of the exogenous LAL. For example, antihistamines can be administered 20, 25, 30, 35 or 40 minutes prior to administration of the exogenous LAL. In one embodiment, the antihistamine administered is diphenhydramine. Any useful antihistamine can be used. Such antihistamines include, without limitation, clemastine, doxylamine, loratidine, desloratidine, fexofenadine, peniramine, cetirizine, evastin, promethazine, chlorpheniramine, levocetirizine, olopatadine, buetapin , Meclizin, dimenhydrinate, embramin, dimethidene and dexchloropheniramine.

In one embodiment, the antihistamine is administered at a dose of about 0.1 mg to about 10 mg per kilogram of body weight. In one embodiment, the antihistamine is administered at a dose of about 1 mg to about 5 mg per kilogram of body weight. For example, the dose may be 1 mg, 2 mg, 3 mg, 4 mg or 5 mg per kilogram of body weight. Antihistamine can be administered by any useful method. In one embodiment, the antihistamine is administered intravenously. In another embodiment, the antihistamine is administered in a pharmaceutically acceptable capsule.

In another embodiment, for intravenous infusion, the likelihood of hypersensitivity reactions may be reduced by administering the infusion using a ramp-up protocol. In this context, the ramp-up protocol refers to slowly increasing the rate of infusion over the course of the infusion to desensitize the patient for infusion of the medicament.

For example, antihistamines, corticosteroids, sirolimus, boclosporin, cyclosporin, but not limited to, prior to, during or after exogenous LAL administration, if a hypersensitivity or detrimental immune response is expected or experienced by the patient. , Methotrexate, IL-2 receptor related antibody, T-cell receptor related antibody, TNF-alpha related antibody or fusion protein (eg, Rifliximab, Etanelcept or adalimumab), CTLA-4-Ig (eg For example, Avacept), an immunosuppressive agent such as an anti-OX-40 antibody may also be administered.

The invention also encompasses treatment involving the administration of an exogenous LAL-containing composition in combination with one or more cholesterol lowering agents (eg, HMG-CoA reductase inhibitors). Non-limiting examples of such agents include: atorvastatin (Lipitor® and Torvast®), fluvastatin (Lescol®), lovastatin (Mevacor®) , Altocor (registered trademark), Altoprev (registered trademark), pitavastatin (Livalo (registered trademark), Pitava (registered trademark)), pravastatin (Pravachol (registered trademark), Selektine (registered trademark), Lipostat (registered trademark)) ), Rosuvastatin (Crestor®) and simvastatin (Zocor®, Lipex®).

Exogenous LAL Effect

The present invention provides for the correction or normalization of disease-related symptoms after treatment with exogenous LAL. Clinical progress in response to exogenous LAL (ie, improvement of condition) can be monitored by any useful method or process.

In some embodiments, administration of the exogenous LAL is sufficient to achieve a Cmax of about 200 ng / ml to about 1,500 ng / ml. In some embodiments, administration of the exogenous LAL is sufficient to achieve a Cmax of about 200 ng / ml to about 1,000 ng / ml. In some embodiments, administration of the exogenous LAL is sufficient to achieve a Cmax of about 200 ng / ml to about 800 ng / ml. In some embodiments, the administration of the exogenous LAL is about 200ng / ml, about 300ng / ml, about 400ng / ml, about 500ng / ml, about 600ng / ml, about 700ng / ml, about 800ng / ml, about 900ng / ml, It is sufficient to achieve a Cmax of about 1,000 ng / ml, about 1,250 ng / ml or about 1,500 ng / ml. In some embodiments, Cmax is reached during infusion.

In some embodiments, the administration of exogenous LAL is sufficient to achieve less than 40 minutes LAL half-life (t _1/2). In some embodiments, the administration of exogenous LAL is sufficient to achieve less than 30 minutes LAL half-life (t _1/2). In some embodiments, the administration of exogenous LAL is sufficient to achieve less than 20 minutes LAL half-life (t _1/2). In some embodiments, the administration of exogenous LAL is sufficient to achieve less than 15 minutes LAL half-life (t _1/2). In some embodiments, the administration of exogenous LAL is sufficient to achieve less than 10 minutes LAL half-life (t _1/2). In some embodiments, the administration of the exogenous LAL is less than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 minutes. it is sufficient to achieve a LAL half-life (t _1/2).

In some embodiments, the exogenous LAL increases LAL activity in the patient. LAL activity can be increased, for example, in the liver, spleen, lymph nodes, aorta, peripheral blood leukocytes and / or skin fibroblasts. In some embodiments, LAL activity is measured in extracts of lymphocytes isolated from blood samples.

Exogenous LAL has at least about 1.5, about 2, about 2.5, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 times the activity prior to LAL administration. Can be increased. Exogenous LALs may increase LAL activity by at least about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 times the activity prior to LAL administration. LAL activity is, for example, cholesteryl [1- ¹⁴ C] oleate, triolein (glycerol tri [1- ¹⁴ C] oleate), p -nitropen myristate or 4-MUO (4 Can be evaluated using methods known in the art, including assays using -methylumbeliperyl oleate) substrates.

In one embodiment, organ and tissue volume and characterization are used to determine amelioration of the disease after administration of the exogenous LAL according to the present invention.

In one embodiment, the clinical progression of liver function / injury over time after administration of exogenous LALs is determined by blood transaminase such as asphaltic acid aminotransferase (AST) and / or alanine transaminase (ALT) and / or other Monitored by quantification of biomarkers such as albumin, alkaline phosphatase and bilirubin (direct and total).

In one embodiment, clinical progress is monitored using imaging techniques. For example, and without limitation, the imaging techniques used may be ultrasound, CT scanning, magnetic resonance imaging, and nuclear magnetic resonance spectroscopy.

In some embodiments, administration of the exogenous LAL at the doses described herein is sufficient to restore growth and / or gain weight in human patients. Administration of exogenous LAL may also increase the growth rate in infants or child patients with premature LAL deficiency (ie, weight gain). For example, administration of the exogenous LAL may include at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, of the growth rate / growth rate seen prior to administration, Weight gain can be increased by about 90%, about 100%, about 200%, about 300%, about 400% or about 500%. In some embodiments, administration of the exogenous LAL restores normal growth rate in a child patient with premature LAL deficiency (eg, monthly illness) that is about 1 month old to about 24 months old. "Normal" in this context refers to the growth rate for patients treated as determined by one of skill in the medical sciences.

In one embodiment, for example for WD and CESD or other LAL deficiency, hepatomegaly is significantly reversed by reverting the liver size to a larger size within about 1% to about 60% than normal. "Normal" in this context refers to a liver of normal size for a treated patient as determined by one of ordinary skill in the medical sciences. In one embodiment, the liver size is reduced by about 1% to more than 50% above normal. In other embodiments, the liver size is reduced by about 1% to about 40% more than normal. In one embodiment, the liver size is reduced from about 1% to more than about 30% above normal. In one embodiment, in another embodiment, the liver size is reduced by about 1% to more than about 20% above normal. In other embodiments, the liver size is reduced by about 10% to about 20% more than normal. For example, the liver can be 10%, 11%, 12% 13% 14%, 15%, 16%, 17%, 18%, 19% or 20% larger than normal size. In yet another embodiment, the liver size is reduced to about 0% to about 10% larger than normal. For example, the liver can be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% larger than the normal size of the liver.

Treatment with exogenous LAL can also improve liver function. Thus, in some embodiments, treatment with exogenous LAL is sufficient to restore liver function to normal and / or normalize liver tests. In some embodiments, treatment with an exogenous LAL is, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, and / or at least about 90% It is sufficient to reduce serum levels of liver transaminoses. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of hepatic transaminase by at least about 40%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of hepatic transaminase by at least about 50%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of hepatic transaminase by at least about 60%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of hepatic transaminase by at least about 70%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of hepatic transaminase by at least about 80%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of hepatic transaminase by at least about 90%.

In some embodiments, the hepatic transaminase is alanine aminotransferase (ALT). In one embodiment, administration of the exogenous LAL is sufficient to reduce serum ALT. For example, administration of the exogenous LAL can reduce serum ALT by, for example, at least about 50%, 60%, 70%, 80% or 90%. Serum ALT levels can serve as an indicator of liver damage. Accordingly, the present invention also contemplates a method of reducing liver damage in human patients with LAL deficiency by administering an effective amount of exogenous LAL to reduce serum ALT.

In some embodiments, the liver transaminase is serum asphaltate transaminase (AST). In one embodiment, administration of the exogenous LAL is sufficient to reduce serum AST. For example, administration of the exogenous LAL can reduce serum AST by at least about 50%, 60%, 70%, 80% or 90%, for example. Serum AST levels can serve as an indicator of liver damage. Thus, the present invention also contemplates a method of reducing liver damage in human patients with LAL deficiency by administering an effective amount of exogenous LAL to reduce serum AST.

In some embodiments, treatment with exogenous LAL can reduce serum ferrin levels. Thus, in some embodiments, treatment with exogenous LAL is for example by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to the level prior to treatment. It is enough to reduce serum ferritin. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of ferritin by at least 50%. In yet another embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of ferritin by at least about 60%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of ferritin by at least about 70%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of ferritin by at least about 80%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of ferritin by at least about 90%. In one embodiment, treatment with exogenous LAL is sufficient to reduce serum levels of ferritin by at least about 95%.

In one embodiment, for example, for moon moon disease and CESD or other LAL deficiency, the spleen is significantly reversed by reverting the spleen size to a size greater than about 1% to about 60% above normal. "Normal" in this context refers to the spleen of normal size for the studied patient as determined by one of ordinary skill in the medical sciences. In one embodiment, the spleen size is reduced from about 1% to about 50% above normal. In another embodiment, the spleen size is reduced from about 1% to about 40% above normal. In one embodiment, the spleen size is reduced from about 1% to about 30% above normal. In another embodiment, the spleen size is reduced from about 1% to about 20% above normal. In another embodiment, the spleen size is reduced from about 10% to about 20% above normal. For example, the spleen can be 10%, 11%, 12% 13% 14%, 15%, 16%, 17%, 18%, 19% or 20% larger than normal size. In yet another embodiment, the spleen size is reduced from about 0% to about 10% above normal. For example, the spleen can be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% larger than the normal size of the spleen.

In one embodiment, administration of the exogenous LAL is sufficient to reduce lymphadenitis (ie, enlarged lymph nodes). Thus, in some embodiments, the lymph nodes are significantly reversed by returning the lymph nodes to a larger size within about 1% to about 60% than normal. “Normal” in this context refers to lymph nodes of normal size for the patients studied as determined by one of ordinary skill in the medical sciences. In one embodiment, lymph node size is reduced from about 1% to about 50% above normal. In another embodiment, lymph node size is reduced from about 1% to about 40% above normal. In one embodiment, lymph node size is reduced from about 1% to about 30% above normal. In another embodiment, lymph node size is reduced from about 1% to about 20% above normal. In another embodiment, lymph node size is reduced from about 10% to about 20% above normal. For example, lymph nodes may be 10%, 11%, 12% 13% 14%, 15%, 16%, 17%, 18%, 19% or 20% larger than normal size. In yet another embodiment, lymph node size is reduced from about 0% to about 10% above normal. For example, the lymph nodes may be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% larger than the normal size of the lymph nodes.

In another embodiment, the lipid analysis is performed to monitor the improvement of the disease. For example, lipid analysis can be done to assess the therapeutic effect of exogenous LAL. Lipid analysis can be performed by any useful method, such as, but not limited to, high performance liquid chromatography, gas chromatography, mass spectrometry or thin film chromatography, or any combination thereof considered appropriate by one skilled in the art, It may be performed on a sample (eg, blood sample, liver biopsy sample). In one embodiment, lipid analysis performed according to the present invention demonstrates levels of total cholesterol, triglycerides, low-density lipoproteins, high-density lipoproteins and / or cholesteryl esters.

In one embodiment, for menopause and CESD or other LAL deficiency, lipid analysis of patients treated in accordance with the present invention is in the liver, spleen, intestine, lymph nodes and / or aorta as can be determined by one of ordinary skill in the medical sciences. Normalization of lipid concentrations is shown.

Lipid levels can be assessed using plasma lipid analysis or tissue lipid analysis. In plasma lipid assays, blood plasma can be collected and total plasma free cholesterol levels can be measured using, for example, color analysis by COD-PAP kit (Wako Chemicals), and total plasma triglycerides For example, it can be measured using a triglyceride / GB kit (Boehringer Mannheim), and / or total plasma cholesterol can be determined using the Cholesterol / HP kit (Boehringer Mannheim). In tissue lipid analysis, lipids can be extracted, for example, from liver, spleen and / or small intestine samples (see, eg, Folch et al. J. Biol . Chem 226 : 497-505 (1957)). Use the provided Folch method). Total tissue cholesterol concentration can be measured using, for example, O-phthalaldehyde.

In some embodiments, administration of the exogenous LAL is sufficient to increase nutrient uptake. In one embodiment, administration of the exogenous LAL increases nutrient uptake as measured by the level of serum alpha tocopherol, 25OH vitamin D, serum retinol, didehydroretinol or transthyretin.

In some embodiments, for example, for moonshine and CESD or other LAL deficiency, administration of exogenous LAL is sufficient to increase serum hemoglobin levels (Hb). In one embodiment, hemoglobin levels are increased by at least about 10% or about 20% compared to what was observed before administration with exogenous LAL.

In some embodiments, exogenous LAL can be administered using a method that minimizes side effects. For example, administration of exogenous LALs can minimize the immune response to exogenous LALs.

Exogenous LAL Containing LAL  And pharmaceutical compositions

The present invention includes treating any of the LAL-deficiency related diseases described herein and other diseases not mentioned above that would benefit from treatment. Exogenous LALs used in accordance with the invention include, without limitation, cell cultures (eg, CHO cells, COS cells), bacteria such as E. coli , transgenic animals such as mammals and birds (eg , Chickens, ducks, and turkeys) and in any useful protein expression system, including in plant systems (eg, frogweed and tobacco). One aspect of the present invention is disclosed in US Pat. No. 7,524,626, issued October 3, 2006; US patent application Ser. No. 11 / 973,853, filed October 10, 2007; 11 / 978,360, filed October 29, 2007; And recombinant LSA generated according to US Pat. No. 12 / 319,396, filed Jan. 7, 2009, the disclosures of which are incorporated herein by reference in their entirety. One aspect of the invention is described in Du et al., (2005) Am . J. Hum . Genet . 77 : 1061-1074 and Du et al., (2008) J. Lipid Res ., 49 : 1646-1657 , which relates to recombinant LALs generated as described herein, the disclosures of which are incorporated herein by reference in their entirety. In one useful embodiment, the exogenous LAL is a transgenic bird (eg, a transgenic chicken) according to the method described in, for example, PCT / US2011 / 033699, filed April 23, 2011, which is incorporated by reference in its entirety herein. ) Is generated from the fallopian tube. In some embodiments, recombinant LALs are produced in avian cell lines. In some embodiments, recombinant LALs are produced in mammalian (eg, human) cell lines.

In one embodiment, the exogenous lysosomal acid lipase used according to the present invention contains glycans having substantially N-acetylglucosamine (GlcNAc) and mannose terminated N-linked structures. GlcNAc and mannose terminated glycans on exogenous LALs are specifically recognized and can be internalized by macrophages and fibroblasts. Mannose-6-phosphate (M6P), which is capable of targeting proteins to GlcNAc / mannose receptors expressed on cells related to diseases that can be treated by exogenous LAL administration, is also typically present on exogenous LALs used in accordance with the present invention. do.

Typically, the exogenous LALs of the present invention discussed and disclosed herein are human LALs. In one embodiment, the exogenous LAL has an amino acid sequence provided in Genbank RefSeq NM_000235.2). In one embodiment, the mature exogenous LAL has the following amino acid sequence:

In some embodiments, the exogenous LAL comprises amino acids 1 to 378 of SEQ ID NO: 1, amino acids 3 to 378 of SEQ ID NO: 1, amino acids 6 to 378 of SEQ ID NO: 1 or amino acids 7 to 378 of SEQ ID NO: 1. In some embodiments, the exogenous LAL is at least 2 selected from the group consisting of amino acids 1 to 378 of SEQ ID NO: 1, amino acids 3 to 378 of SEQ ID NO: 1, amino acids 6 to 378 of SEQ ID NO: 1, and amino acids 7 to 378 of SEQ ID NO: 1 Mixtures of two polypeptides. In some embodiments, the exogenous LAL comprises a mixture of polypeptides comprising amino acids 1 to 378 of SEQ ID NO: 1, amino acids 3 to 378 of SEQ ID NO: 1 and polypeptides comprising amino acids 6 to 378 of SEQ ID NO: 1. In some embodiments, the exogenous LAL comprises polypeptides identical to amino acids 1 to 378 of SEQ ID NO: 1, amino acids 3 to 378 of SEQ ID NO: 1, amino acids 6 to 378 of SEQ ID NO: 1 or amino acids 7 to 378 of SEQ ID NO: 1 . In other embodiments, the exogenous LAL comprises at least about 70% of amino acids 1 to 378 of SEQ ID NO: 1, amino acids 3 to 378 of SEQ ID NO: 1, amino acids 6 to 378 of SEQ ID NO: 1 or amino acids 7 to 378 of SEQ ID NO: 1 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical polypeptides. In some embodiments, the exogenous LAL comprises a polypeptide that is a functional fragment of SEQ ID NO: 1 or at least about 70%, about 75%, about 80%, about 85%, about 90%, about a functional fragment of SEQ ID NO: 1 95%, about 96%, about 97%, about 98% or about 99% identical.

In some embodiments the exogenous LAL is a recombinant LAL protein described in PCT / US2011 / 033699, filed April 23, 2011, which is incorporated herein by reference in its entirety.

When amino acid residues are substituted with other amino acid residues having similar chemical properties (e. G., Charge or hydrophobicity), the non-identical amino acid positions are often differentiated by conservative amino acid substitutions and thus do not alter the functional properties of the molecule. If the sequence differs from conservative substitutions, the percent sequence identity can be upregulated to be modified for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. Scoring of conservative substitutions is described, for example, in Meyers & Millers, Computer Applic. Biol. Sci. 4: 11-17 (1988)].

"Comparison window" refers to a segment of contiguous positions, such as about 25 to about 400 positions, or about 50 to 200 positions, or about 100 to 150 positions, which are consecutive positions after the two sequences are optimally aligned. It can be compared with a reference sequence of the same number. Methods for aligning sequences for comparison are well known to those skilled in the art. The optimal alignment of sequences for comparison is, for example, by regional homology algorithms (Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by global alignment algorithms (Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by investigation of similarity methods (Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); Altschul et al., Nucl.Acids Res. 25: 3389 -402 (1997), by computerized implementation of these algorithms (e.g., Wisconsin Genetics Software Group's Wisconsin Genetics software package, GAP, BESTFIT, FASTA, and BLAST), typically using default settings Or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al. (Eds.), 1994). Army of SEQ ID NO: 1 performed using the XBLAST program, score = 50, word length = 3 By acid sequence or its fragment and a greater than 80% it can be obtained the same amino acid sequence.

One example of a useful algorithm implementation is PILEUP. PILEUP makes use of progressive pairwise alignments to make multiple sequence alignments from a group of related sequences. You can also plot the schematic representing the clustering relationship used to create the alignment. PILEUP is described in Feng & Doolittle, J. Mol. Evol. 35: 351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-3 (1989). Many alignment processes begin with bi-alignment of the two most similar sequences, creating a cluster of two aligned sequences. This cluster may then be aligned with the next most relevant sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by simple extension of the bilateral alignment of two individual sequences. A series of such bilateral alignments, including increasingly dissimilar sequences and clusters of sequences in each iteration, produce the final alignment.

In some embodiments, the exogenous LAL polypeptide of the invention comprises variants of wild type sequences. These variants belong to one or more of three classes: substitutional, insertional, or deletional variants. These variants may be naturally occurring alleles or interspecies variants or they may be prepared by site-specific mutagenesis of nucleotides in DNA encoding proteins. Site-specific mutagenesis can be performed using cassettes or other techniques well known in the art for generating PCR mutagenesis or DNA encoding the variants, and then expressing the DNA in the recombinant cell culture. Variant target protein fragments having up to about 100 to 150 amino acid residues may be prepared by in vitro synthesis using established techniques. Conservative substitution tables that provide functionally similar amino acids are well known in the art (Henikoff & Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919 (1992)).

Amino acid substitutions are typically single residues. While significantly longer insertions can be tolerated, insertions can usually be along about 1 to about 20 amino acids. Deletion is in the range of about 1 to 20 residues, and in some cases the deletion may be much longer. Substitutions, deletions and insertions or any combination thereof can be used to arrive at the final derivative.

In some embodiments, the exogenous LAL has a specific activity of at least about 100 U / mg. In some embodiments, the exogenous LAL has a specific activity of at least about 200 U / mg. In some embodiments, the exogenous LAL has a specific activity of at least about 250 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 100 to about 1,000 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 100 to about 500 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 100 to about 350 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 200 to about 350 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 250 to about 350 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 250 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 275 U / mg. In some embodiments, the exogenous LAL has a specific activity of about 300 U / mg. Human LAL has six potential sites in its amino acid sequence for N-linked glycosylation: Asn36, Asn72, Asn101, Asn161, Asn273 and Asn321 as shown in SEQ ID NO: 1. In some embodiments, at least one, two, three, four or five of the N-linked glycosylation sites are glycosylated. In some embodiments all six glycosylation sites are glycosylated. In some embodiments, Asn36, Asn101, Asn161, Asn273 and Asn321 are glycosylated. In some embodiments, Asn36, Asn101, Asn161, Asn273 and Asn321 are glycosylated and Asn72 is not glycosylated. In some embodiments, the N-glycan structure comprises bi-, tri- and tetraantennary structures with N-acetylglucosamine (GlcNAc), mannose, and / or mannose-6-phosphate (M6P). In some embodiments, the exogenous LAL comprises M6P-modified N-glycans at Asn101, Asn161 and Asn273. In some embodiments, the exogenous LAL does not comprise O-linked glycans. In some embodiments, the exogenous LAL does not comprise sialic acid. In some embodiments, the exogenous LAL has a glycosylation pattern as described in PCT / US2011 / 033699, filed April 23, 2011, which is incorporated by reference in its entirety.

In some embodiments, the molecular weight of the exogenous LAL is about 55 kD.

In certain embodiments, the subject can be treated with a nucleic acid molecule that encodes an exogenous LAL, for example, in a vector. Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg or 30 to 300 μg DNA per patient. Doses for infectious viral vectors vary from virion per dose to 10 to 100 or more.

In some embodiments of the invention, the exogenous LAL is administered in a therapeutic method comprising: (1) an implantable host cell, eg, a vector, having a nucleic acid expressing the LAL or an active fragment, variant or derivative thereof Transforming or transfecting; And (2) transplanting the transformed host cell into a mammal. In some embodiments of the invention, the implantable host cell is removed from the mammal, transiently cultured, transformed or transfected with an isolated nucleic acid encoding an exogenous LAL and transplanted back into the same mammal from which it has been removed. . The cells may be removed from the same site implanted, although this is not necessary. In such embodiments, sometimes known as ex vivo gene therapy, one may provide a continuous supply of exogenous LAL polypeptide for a limited time period.

While it is possible to be administered in the original form to recombinant LAL, the therapeutic protein provided for the present invention, it is preferred to administer the therapeutic protein as part of a pharmaceutical preparation.

The present invention therefore provides a pharmaceutical preparation comprising an algal-derived glycosylated therapeutic protein or a pharmaceutically acceptable derivative thereof in combination with one or more pharmaceutically acceptable carriers thereof and optionally other therapeutic and / or prophylactic components. And methods of administering such pharmaceutical preparations. The present invention also provides pharmaceutical preparations comprising a mammalian-derived glycosylated therapeutic protein or a pharmaceutically acceptable derivative thereof in combination with one or more pharmaceutically acceptable carriers and optionally other therapeutic and / or prophylactic components. Methods of administering water and such pharmaceutical preparations are provided.

The carrier (s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and must not be detrimental to its receptor. Methods of treatment (eg, amount of pharmaceutical protein administered, frequency of administration, and duration of treatment) of patients using the pharmaceutical compositions of the invention can be determined using standard methods known to those skilled in the art.

Pharmaceutical preparations include those suitable for oral, rectal, nasal, topical (including oral and sublingual), vaginal or parenteral. Pharmaceutical preparations include those suitable for administration by injection, including intramuscular, subcutaneous and intravenous administration. Pharmaceutical preparations also include those for administration by inhalation or inhalation. Formulations may, if appropriate, be conveniently presented in separate dosage units and may be prepared by any of the methods well known in the art of pharmacy. Methods of producing pharmaceutical formulations typically include causing a therapeutic protein associated with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical preparations suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of active ingredient; As a powder or granules; As a solution; As a suspension; Or as an emulsion. The active ingredient can also be presented as a bolus, soft or paste. Tablets and capsules for oral administration may contain conventional excipients such as binders, fillers, lubricants, disintegrants or wetting agents. Tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid formulations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

Therapeutic proteins of the invention may also be formulated for parenteral administration (eg, by injection, eg, bolus injection or continuous infusion), and may be in unit dosage form, pre-filled syringe, small dose infusion in ampoules Or in a multi-dose container with added preservative. Therapeutic proteins can be injected, for example, by subcutaneous injection, intramuscular injection and intravenous (IV) infusion or injection. In one embodiment, the exogenous LAL is administered intravenously by IV infusion by any useful method. In one embodiment, the exogenous LAL can be administered by intravenous infusion via the peripheral line. In another example, the exogenous LAL can be administered by intravenous infusion via a centrally inserted central catheter. In another example, the exogenous LAL may be administered by intravenous infusion facilitated by a mobile injector attached to the venous vascular access port. In one embodiment of intravenous infusion, the medicament is determined by one of skill in the art and administered over a period of 1 to 8 hours depending on the amount of medicament injected and the patient's previous infusion-related response history. In another embodiment, the exogenous LAL is administered intravenously by IV injection. In other embodiments, the exogenous LAL can be administered via intraperitoneal injection. In another embodiment, the exogenous LAL is administered via a pharmaceutically acceptable capsule of the therapeutic protein. For example, the capsule can be an enteric-coated gelatin capsule.

In some embodiments, the therapeutic protein is administered by infusion and the infusion can occur over an extended time period, such as 30 minutes to 10 hours. Thus, the infusion can occur, for example, over a time period of about 1 hour, about 2 hours, about 3 hours, about 4 hours or about 5 hours. Injection can also occur at various rates. Thus, for example, the infusion rate can be from about 1 ml per hour to about 20 ml per hour. In some embodiments, the infusion rate may be about 5 ml to about 10 ml per hour. In one embodiment, the infusion rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ml per hour. to be. In one embodiment, the infusion rate is 0.1-5 mg / kg / hour. In one embodiment, the infusion rate is about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 1.5, about 2.0, or about 3 mg / kg / hour.

The therapeutic protein may take this form as a suspension, solution or emulsion in an oily or aqueous vehicle and may contain a formulation agent such as suspending, stabilizing and / or dispersing agents. The therapeutic protein may be in powder form obtained by aseptic separation of sterile solids or by lyophilization from solution for constitution with a suitable vehicle, eg sterile, pyrogen-free water, before use.

For topical administration to the skin, the therapeutic protein may be formulated as an ointment, cream or lotion or as a transdermal patch. Ointments and creams may be formulated with an aqueous base or an oily base, for example by the addition of suitable thickening and / or gelling agents. Lotions can be formulated with an aqueous or oily base and will generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or coloring agents.

Formulations suitable for oral topical administration include lozenges comprising the active ingredient in a scented base, usually sucrose and acacia or tragacanth; Pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; And mouthwashes comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical formulations suitable for rectal administration (carriers are solid) are most preferably represented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and suppositories can be conveniently formed by a mixture of soft or molten carrier (s) with the active compound, followed by cooling and molding in the mold.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing such carriers which are known in the art as further suitable for the active ingredient.

For intranasal administration, the therapeutic proteins of the invention can be used as liquid sprays or dispersible powders or in drop form.

Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.

For administration by inhalation, the therapeutic protein according to the invention may conveniently be delivered from a blower, nebulizer or other convenient means of delivering a pressurized package or aerosol spray. Pressurized packs may include suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for delivering a metered amount.

For administration by inhalation or inhalation, the therapeutic protein according to the invention can take the form of a dry powder composition, for example a mixture of a powder and a suitable powder base such as lactose or starch. The powder composition may be provided in unit dosage form, eg a capsule or cartridge or a gelatin or blister pack, for example where the powder may be administered with the aid of an inhaler or blower. If desired, the formulations described above suitable for providing sustained release of the active ingredient can be used.

The pharmaceutical compositions according to the invention may also contain other active ingredients such as antimicrobial agents or preservatives.

In some embodiments, the concentration of exogenous LAL in the pharmaceutical composition is about 0.5 to about 10 mg / ml. In some embodiments, the concentration of LAL is about 1 to about 5 mg / ml. In some embodiments, the concentration of LAL is about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5 or about 7.5 mg / ml. to be.

In some embodiments, the pharmaceutical composition comprising the exogenous LAL further comprises a buffer. Representative buffers include acetate, phosphate, citrate and glutamate buffers. Representative buffers also include lithium citrate, sodium citrate, potassium citrate, calcium citrate, lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, maleic acid Potassium, calcium maleate, lithium tartarate, sodium tartarate, potassium tartarate, calcium tartarate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate and mixtures thereof. In some embodiments, the buffer is trisodium citrate dihydrate. In some embodiments, the buffer is citric acid monohydrate. In some embodiments, the pharmaceutical preparation comprises trisodium citrate dehydrate and citric acid monohydrate.

In some embodiments, the pharmaceutical composition comprising the exogenous LAL further comprises a stabilizer. Representative stabilizers include albumin, trehalose, sugars, amino acids, polyols, cyclodextrins, salts such as sodium chloride, magnesium chloride and calcium chloride, lyoprotectants and mixtures thereof. In some embodiments, the pharmaceutical composition comprises human serum albumin.

In specific embodiments, recombinant human LALs produced as disclosed herein are used in pharmaceutical preparations, with each milliliter of exogenous LAL (eg, 2 mg LAL), trisodium citrate dehydrate (eg, , 13.7 mg), citric acid monohydrate (eg 1.57 mg) and human serum albumin (eg 10 mg) and are prepared at an acidic pH such as 5.9 ± 0.1. The present invention includes any route of administration that facilitates absorption of exogenous LAL into appropriate organs and crude lysosomes.

Example

The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims.

Example  One

Recombination LAL Coarseness by administration of LAL  Deficiency ( Moon cake Treatment of

Male infants were hospitalized at 15 weeks of age due to poor weight gain after birth (birth weight, 3.88 kg). Infants had vomiting, eating problems, poor nutrition, diarrhea, increased bloating and anemia. The patient was diagnosed with moon balm.

In the initial physiological test, the patient weighed 5.62 kg and he is located below the fifth percentile of age weight. Patients did not gain weight over the next 4 weeks from the initial examination at 15 weeks of age and before the initial infusion at 19 weeks of age. The estimated growth rate was calculated to be below the first percentile over age weight. The abdomen was markedly swollen with significant hepatomegaly and splenomegaly. Abdominal ultrasound and CT scans showed symmetric enlarged adrenal glands on both sides with hepatomegaly and calcification. As with the asphaltate transaminase (AST) at 216U / L (normal 10-45U / L), the level of serum alanine transaminase (ALT) was raised to 119U / L (normal 10-50U / L). Prior to initiation of treatment, the serum marker ferritin was approximately 1,500 μg / L (usually 7-144 μg / L). The patient had persistent anemia before treatment with hemoglobin values ranging from 7.2 to 8.3 g / dL.

At 19 weeks of age, IV infusion of rhLAL (SBC-102) was initiated once weekly at an initial dose of 0.2 mg / kg. In order to counteract the potential infusion reaction, patients were pretreated with 1 mg / kg of diphenhydramine at approximately 90 minutes prior to infusion of SBC-102. Infusion duration was approximately 4 hours. The infusion was well tolerated and the patient experienced no side effects or infusion related reactions.

Seven days after the initial infusion, the patient received a second injection. Patients were dosed with 0.3 mg / kg SBC-102 for approximately 4 hours and withstood the infusion without showing any signs of side effects.

Within two weeks of treatment, patients generally showed a significant improvement in well-being, including increased alertness and responsiveness. Diarrhea and vomiting stabilized. Patients began to gain weight and showed a marked decrease in serum transaminase (eg, AST and ALT), essentially normal levels (FIGS. 1A and 1B). Patient growth rates normalized rapidly (FIGS. 3 and 4). Abdominal distension corresponding to a decrease in abdominal circumference was reduced. Liver function tests showed continuous improvement (FIGS. 1A and 1B).

At the third visit, the patient received SBC-102 at 0.5 mg / kg. Injection continued and was well tolerated. The clinical condition showed a continuous improvement with weight gain of 150 g on day 7 and 1.5 cm increase around the arm after initiation of treatment (FIGS. 3 and 4). Liver tests were stable, hemoglobin levels increased (10-11 g / dL), and ferritin levels continued to decrease (FIG. 2). Alkaline phosphatase was in the low normal range 137 U / L (normal 110-300 U / L) before treatment and increased with treatment (204 U / L). This effect by SBC-102 administration is consistent with observations made in preclinical disease models.

Beginning with the fourth infusion, patients began receiving a dose of 1.0 mg / kg weekly. Two months after the start of treatment, patient growth was substantially improved at an estimated growth rate close to the 95th percentile. This increase in growth resulted in 1.25 kg or 2.79 lbs at 7.21 kg on day 63, which was placed in the 30th percentile of age weight (FIGS. 3 and 4). Both AST and ALT levels decreased rapidly after the first injection.

Three months after treatment, AST and ALT were normal. In addition to improving liver function, a significant decrease in ferritin was also observed (FIG. 2).

Over four months of treatment, the patient's GI symptoms were resolved and the patient's nutritional status was excellent. The patient gained weight continuously (FIGS. 3 and 4) and demonstrated physical signs of normal healthy infants. The patient withstood the infusion continuously without exhibiting any infusion reaction or other side effects. The patient received the 21st dose of 1.0 mg / kg as a hospital patient.

Example  2

Precocious LAL  Study design for deficiency

SBC-102, a rhLAL produced from transgenic galus, is administered by IV infusion weekly. A study is designed to evaluate the safety, tolerability and efficacy of the two-dose regimen of SBC-102 administered as a weekly IV infusion. As such, key outcome variables in this study determine the safety and tolerability of SBC-102 in children with growth failure due to LAL deficiency and include: vital signs and physical examination findings; Clinical laboratory testing; Anti-drug antibody testing; And use of concurrent agents. Given that growth failure is a universal clinical feature of LAL deficiency / month phenotype, successful therapies for this disorder should be able to handle the growth failure seen in children with LAL deficiency. Variables directly related to child growth and nutritional status are evaluated as second or preliminary subjects: for example, increasing rate of growth with respect to body weight; Weight gain; And linear growth rate. This study also included pharmacodynamic biomarkers; Liver and spleen size; Lymphadenitis; Hemoglobin and platelets; Laboratory evaluation of liver function and nutrition; Investigate the effects of SBC-102 on abdominal circumference, brachial circumference and head circumference. The study also describes the preliminary pharmacokinetics of SBC-102 in children with growth failure due to LAL deficiency, including plasma Cmax and estimated clearance.

Subjects are enrolled in two consecutive cohorts of the same size (4 subjects each). Doses are staggered within each cohort and between cohorts, starting dosing with the first subject in a lower dose cohort (cohort 1; starting at dose 0.35 mg · kg ⁻¹ ). The dose of additional subjects in Cohort 1 and the commencement of dosing in higher dose cohorts (Cohort 2; starting at dose ¹ mg · kg ⁻¹ ) is based on acceptable safety and tolerability in the preceding subjects.

Cohort  One

The first four subjects enrolled in the study make up Cohort 1. In this cohort, the first subject receives a single dose of SBC-102 0.35 mg · kg ⁻¹ , and if a continuous dose based on a safety review is approved at least 24 hours after dosing, the subject will receive SBC- 1 week later. A second dose of 102 0.35 mg · kg ⁻¹ is received. Determines the possibility whether or not acceptable for the start of the first cohort, and whether the medication in a subject that other increase in ^1-piheom after receiving the second dose of the SBC-body 102, the capacity of the first to-be-heonche 1㎎ · ㎏ All available safety data is reviewed at the time of entry. Dosing of the other three subjects from Cohort 1 proceeds in a similar manner. Safety review in 0.35㎎ · ㎏ ^-1 1㎎ · ㎏ ^- does not guarantee that the increase in the capacity of a subject ^1, if it is to continue the process with the starting dose considered safe for a subject, a subject is 0.35㎎ · ㎏ You will continue to receive ^-1 doses. If any cohort of subjects is 1 shows the inadequate response to the treatment after at least 4 times the capacity of the ^{SBC-102 1㎎ · ㎏ -1,} 3㎎ · ㎏ - an additional capacity increase is considered to be ^one.

Cohort  2

Initiation of dosing in Cohort 2 occurred after Cohort 1 was fully registered, and safety was reviewed for at least two subjects who received two or more doses of 1 mg · kg ⁻¹ SBC-102 in Cohort 1. The last four subjects who entered the study were enrolled and dosed in Cohort 2. The first cohort in a subject is SBC-102 1㎎ · ㎏ ^- if approved for continued dosing based on receive a single dose of ^1, the safety review through at least 24 hours after dosing, a subject is SBC- after 1 week 102 receives a second dose of ¹ mg · kg ⁻¹ . After receiving the second dose of piheom SBC-body 102, and review all possibilities available safety data following acceptance of: 3㎎ · ㎏ for the first a subject ^- increase the capacity to ¹ and the other blood of a cohort 2 Start dosing of subjects. Dosing of three other subjects from Cohort 2 is performed in a similar manner with safety review. Safety reviewers 3㎎ · ㎏ in 1㎎ · ㎏ ^{-1 -} does not approve the increase of the capacity of a subject ^1, if it is to continue the process from the start considered a safe dose to a subject, a subject is 1㎎ · ㎏ You will continue to receive ^-1 doses. If a starting dose of ¹ mg · kg ⁻¹ is not well tolerated by the subject, a reduced dose of 0.35 mg · kg ⁻¹ may be considered.

The study consisted of approximately 22 schedule visits: visit 1 (screening), visit 2 (baseline assessment, initiation of study drug) to visit 21 (weekly administration of study drug), visit 22 (end of follow-up study). Given the severity and life-threatening nature of precocious growth failure due to LAL deficiency, these subjects are likely to be hospitalized.

Target populations for this study are boys and girls with growth failure due to LAL deficiency. Subjects may participate in this study if the following criteria are met: (1) Any course of study conducted that understands the complete nature and purpose of the study, including the possible risks and adverse effects of the subject's parent or legal guardian. Provide written informed consent / permissions; (2) boys or girls with a recorded record of reduced LAL activity or a molecular genetic test confirming the diagnosis of LAL deficiency compared to the normal range of the laboratory performing the assay; And (3) growth failure initiated 6 months of age.

safety

Key safety endpoints include the occurrence of adverse events (AEs) and infusion related reactions (IRR); Changes from baseline in vital signs (blood pressure, heart rate, respiratory rate and body temperature), physical examination points and clinical laboratory tests (CBC / hematology, serum chemistry and urinalysis); Use of concurrent medications / treatments; And characterization of the anti-SBC-102 antibody (ADA) including seroconversion rate, time to seroconversion, median and time to peak immunoglobulin G (IgG) ADA titer and peak IgG ADA titer do.

efficacy

Efficacy endpoints include (1) changes and / or percent change from baseline in liver and spleen size (by ultrasound) and liver and spleen volume and fat content (by magnetic resonance imaging [MRI]); And (2) changes from baseline in serum transaminase, serum lipids (total cholesterol, triglycerides, high density lipoproteins [HDL], and low density lipoproteins [LDL]), hemoglobin and platelet counts. Growth variables, including changes from baseline in percentiles and z-scores, are also assessed for subjects under 18 years of age. These growth variables are based on the Centers for Disease Control (CDC) growth chart and include weight-for-age (WFA), weight-for-length (WFL) and age-length (length- for-age (LFA) and head circumference-for-age (HCFA) and WFA, age-for-age (SFA) in subjects under 30 months of age; Height) and weight-for-stature (WFS) in subjects from 36 months of age to 18 years of age, as well as the corresponding growth status indicators, consumable and stunted growth below substandard weight in all subjects. Include.

Example  3

Precocious LAL  Physical assessment of patients with deficiency

Patients who are clinically stable to tolerate general anesthesia should consider central venous insertion for prolonged vascular access. In subjects undergoing general anesthesia and / or sedation for other procedures, a reference abdominal magnetic resonance imaging (MRI) scan is considered. In new course events that require general anesthesia and / or sedation, follow-up MRIs are considered if not earlier than 3 months after the first injection. Anthropometric measurements (weight, height, abdomen circumference, upper arm circumference and head circumference) are measured. Perform a general physical examination. Perform a complete physical examination. Examinations include the general appearance of the subject, skin, head, eyes, ears, nose and neck, heart, lungs, abdomen, limbs / joints, and nerve conditions. All physical examinations also include:

Liver size: Creates a clinical assessment of the size of the liver (centimeters below detectable / undetectable and costal margin), regularity (smooth / nodular) and sensitivity (stingy / not tingling).

Spleen Size: Makes clinical assessment of spleen size (sensable / undetectable and centimeters below the leading edge), regularity (smooth / nodular) and sensitivity (smooth / soft).

Lymphadenitis : An assessment of the size, location and any detectable characteristic of the lymph nodes is made. Areas examined include the following: head (back head, front anterior, posterior ear, submandibular, submandibular gland), cervix, collarbone, armpits and inguinal area. Any extended node is characterized by stinging or not stinging.

Photos: A digital photograph (full length and abdominal close-up) of the subject is taken at supine position.

Liver / splenic ultrasound and MRI

Abdominal ultrasound is performed to measure liver and spleen size. Abdominal MRI can provide better quantification of liver and spleen volumes and is considered at baseline and at least 3 months visit after the first injection.

Vital signs

Vital signs include pulse rate, respiratory rate, heart contraction and diastolic blood pressure and central body temperature (rectal or oral). Pulse rate and blood pressure are evaluated after subject is supine. Vital signs are measured at all study visits. On the dosing day, vital signs are recorded before infusion, every 15 minutes (± 10) during the infusion and 2 hours after the infusion, then every 30 minutes (± 15) 2 to 4 hours after the infusion is complete.

Example  4

Laboratory evaluation

The following laboratory evaluations are performed as diagnostic tests and efficacy:

1) CBC / hematology: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean blood cell hemoglobin concentration (MCHC) Peripheral smear for testing of platelet count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, cell morphology

2) Chemical panel: glucose, urea nitrogen, creatinine, sodium, potassium, chloride, calcium (total and ionized), magnesium, inorganic phosphorus, total protein, lactate dehydrogenase

3) Liver function test: AST / serum glutamic oxaloacetic transaminase (SGOT), ALT / serum glutamic pyruvic transaminase (SGTG), alkaline phosphatase, gamma-glutamyl Transpeptidase (gamma-glutamyl transpeptidase (GGTP)), albumin, bilirubin (direct, whole)

4) Anti-Drug Antibodies: Anti-SBC-102 Antibodies

5) Urine test: pH, glucose, ketones, blood, protein, nitrate

6) Coagulation Studies: Leukocytes (microscopic examination may be done if blood, nitrates and / or leukocytes are abnormal)

7) Study Nutrition Assessment: Serum Alpha Tocopherol: Cholesterol Ratio, 25OH Vitamin D, Serum Retinol, Didehydroretinol, Transthyretin, Serum Ferritin.

8) Lipid Panel: Total Cholesterol, Triglycerides, HDL, LDL

9) general profile

DNA sequences comprising both protein coding sequences and sequences controlling gene transcription, messenger ribonucleic acid (mRNA) stability, and potency of protein translation that can be identified include:

1. Lysosomal Acid Lipase (LIPA Gene)

2. Genetic code for other proteins involved in lipid biology that may contribute to and / or modify the disease phenotype of LAL deficiency, eg ABCA1

3. Genes That Can Alter Susceptibility to Any SBC-102

10) Pharmacokinetic Evaluation

To reduce the risk of clinic anemia, PK sampling can be limited. In order of importance, samples are collected to obtain the following variables: 1) Cmax and 2) CL estimation. On day 0 (dose 1) and day 105 (dose 16), sampling for the determination of SBC-102 serum levels is collected. In all subjects, samples were administered prior to dosing (within 30 minutes of dosing); 90 (± 5) minutes after start of infusion; And 110 (± 5) minutes after start of infusion. All PD samples should be taken at the same time as the assessment of vital signs prior to cuff inflation for BP assessment on non-injected arms. PK samples are taken at different time points at least 5 minutes after cuff contraction.

Example  5

Dose Preparation and Injection

SBC-102 is provided as a clear liquid in a single dose 10 ml glass vial. The solution (10.5 ml total, including 5% overfill) has a concentration of 2 mg · ml ⁻¹ . All SBC-102 vials were stored at a controlled temperature of 2 ° C. to 8 ° C. Freeze vials and protect from light during storage. Syringes containing SBC-102 diluted in 0.9% saline were prepared just prior to injection. When a syringe of SBC-102 was prepared beforehand, the dilution solution was labeled and used within 4 hours of preparation.

The body weight of patients recorded before dosing at the infusion morning and rounded to near 0.1 kg was used to calculate the SBC-102 volume for each infusion. The total infusion volume used in the study is based on the dosage regimes shown in Table 2.

Volume Injection volume 0.35mg / kg 10 ml 1mg / kg 10 ml 3 mg / kg 20 ml

Dose preparation and administration should be performed using sterile pyrogen-free disposable materials including, but not limited to, syringes, needles, transport tubing, and stoppers.

The infusion rate on the flow-control device should be set to administer the entire volume over approximately 120 minutes as shown in Table 3.

Volume Injection speed time Injection rate per minute Injection rate / kg / hour 0.35mg / kg 5 ml 0.083 ml 0.175 mg / kg / hour 1mg / kg 5 ml 0.083 ml 0.5 mg / kg / hour 3 mg / kg 10 ml 0.167 ml 1.5 mg / kg / hour

Example  6

Side Effect( AE )

In subjects who have experienced AEs or infusion related reactions (IRRs) with clinically significant cardiovascular, respiratory or other effects, the infusion should be discontinued and the subject should be institutional for the management of severe infusion reactions in children under 2 years of age. Treatment with hypersensitivity reactions should be done in accordance with the guidelines. This may include intravenous antihistamines, corticosteroids and epinephrine, if desired. For related biological products, the majority of delayed IRR occurs more than 24 hours after injection. Symptoms include arthralgia, myalgia, influenza-like symptoms, headache, fatigue and rash or hives. The delayed response may be treated with analgesics or antihistamines as clinically indicated. IRR is classified as acute (occurring within 24 hours of injection) or delayed (occurring 1-6 days after injection). Agents and apparatus for the treatment of hypersensitivity reactions should be readily available in case of unexpected, severe hypersensitivity reactions. These feeds include, but are not limited to, oxygen, acetaminophen, antihistamines (eg, diphenhydramine, parenteral and PO), corticosteroids, epinephrine, and cardiopulmonary resuscitation devices. In similar biological products, the most acute IRR occurs within 24 hours of infusion (Cerezyme®, VPRIV®, Fabrazyme® prescription information). Possible signs of acute IRR can be categorized as follows: Mild to moderate IRR: hyperemia, redness, fever and / or chills, nausea, pruritus, urticaria, gastro-intestinal symptoms (vomiting, diarrhea, abdominal cramps). Mild response is defined as self-limiting to resolve the response spontaneously after a temporary pause or a decrease in the rate of infusion. Moderate reactions are not resolved by simple measurements and are defined as responses that require prolonged observation and discontinuation of treatment. Severe IRR involves chest pain, shortness of breath, wheezing, wheezing, low or high blood pressure, respiratory arrest, apnea, shortness of breath, bradycardia or tachycardia. If any of the above signs and symptoms are observed during infusion and the subject remains hemodynamically stable, the infusion rate may be slowed down (half the rate given at the onset of the event, for example 10 ml · hr ⁻¹ to 5 Ml · hr ⁻¹ ) the injection time is extended. Once the event is resolved, the infusion should last at least 30 minutes at the reduced rate before the rate is increased to 75% of the original rate on the infusion schedule. If the subject continues to show signs of hypersensitivity, an IM or slow IV dose of antihistamine should be administered for the hypersensitivity response in accordance with institutional guidelines for infusion response management in children under 2 years of age.

Example  7

Late LAL  For human patients with deficiency rhLAL Dosing

The main purpose of the study was to assess the safety and tolerability of SBC-102 in patients with hepatic dysfunction due to late LAL deficiency (active signs, physical examination, clinical laboratory tests, immunogenicity tests, side effects assessment, concurrent treatment). . The second purpose is to characterize the pharmacokinetics of SBC-102 delivered by IV infusion after single and multiple doses (before injection and days 0 and 21 after injection). Inclusion criteria for late LAL deficiency subjects are as follows:

1. The patient understands the complete nature and purpose of this study, including possible risks and adverse effects, is willing to follow all research procedures, can follow and provide informed consent;

2. Male or female patient over 18 years old and under 65 years old;

3. Recorded results of molecular genetic testing confirming a record of reduced LAL activity or a diagnosis of LAL deficiency compared to the normal range of the laboratory performing the assay;

4. Liver related evidence based on clinical manifestations (hepatomegaly) and / or laboratory test results (ALT or AST ≧ 1.5 × ULN);

5. If statin or ezetimibe is present, the patient must have a stable dose for at least 4 weeks prior to screening;

6. All women should be negative for serum pregnancy tests at screening and not breastfeeding; And

7. Probable female patients must give consent for the use of highly effective and approved contraceptive method (s) to continue the study and must continue for 30 days after the last dose.

Clinical evaluation, including physical examination, urinalysis, and clinical chemistry analyzes, CBC / hematology, acute phase reactants, coagulation studies, 12-lead ECG, wherein -SBC-102 antibody and _{PK Cmax, AUC inf, T 1} /2 , Cl and V _SS are obtained.

Patients receive 0.35 mg · kg ⁻¹ , ¹ mg · kg ⁻¹ or 3 mg · kg ⁻¹ of LAL once weekly via intravenous (IV) infusion over 2 hours. Dosing to the first subject is monitored for at least 24 hours prior to proceeding to other subjects in the cohort. Each subject remains inpatient for 24 hours after the first injection of SBC-102. Subjects continue with an additional 3 doses of IV infusion once per week of SBC-102 doses, provided that tolerability and safety remain acceptable.

Pharmacokinetics

PK data is analyzed using all subjects entering the study who received at least one dose of study medication, except for any data points that may be affected by major protocol deviations. PK analysis is performed using a 1 compartment injection model. It obtained the following PK parameters, which are provided by cohort (Cmax, AUC _inf, T _1/2, Cl and V _SS). Single and multiple dose PK variables are compared using second and sixth visit data.

Suggested increases between doses in this study can be 3, 4, 5 or 6-fold, which would allow an assessment of initial safety, tolerability and pharmacokinetics in humans over a 6-fold range of dose on a mg · kg ⁻¹ basis. Can be. In an appropriate preclinical rat model, it may compare the 1㎎ · ㎏ ^-1 1 once every 1 weeks, 3㎎ · ㎏ ^-1 every other week and 5㎎ · ㎏ ^-1 1 meeting pharmacodynamic effect of each week. Thus, 3 ㎎ · ㎏ ^-1 per 1 ju is not expected to need the capacity of more than once, 3 ㎎ · ㎏ ^- capacity of greater than ^1, for example 4, 5, 6, 7, 8, 9, or 10㎎ · ㎏ ^-1 can be considered depending on the severity of the mass.

Research design

Given the rareness of patients with this disease, the number targeted for this study is 9 evaluable subjects. Subjects are enrolled in three consecutive cohorts of 3 subjects per cohort. Dosing first begins with the subject assigned to Cohort 1, then the subject assigned to Cohort 2 and then to the subject assigned to Cohort 3.

Cohort  One

Three subjects received an IV infusion of 0.35 mg · kg ⁻¹ SBC-102. Dosing to the first subject and monitoring for tolerability for at least 24 hours prior to proceeding to other subjects in the cohort. Each subject remains an inpatient for 24 hours after the first injection of SBC-102. Subjects continued with an additional three IV infusions of 0.35 mg · kg ⁻¹ , provided that tolerability and safety remained acceptable.

Cohort  2

Three subjects received an IV infusion of SBC-102 at ¹ mg · kg ⁻¹ . Dosing to the first subject in Cohort 2 and monitoring for tolerability for at least 24 hours prior to proceeding to the other two subjects in Cohort. Each subject remains an inpatient for 24 hours after the first injection of SBC-102. Subjects continued with three additional IV infusions of ¹ mg · kg ⁻¹ , provided that tolerability and safety remained acceptable.

Cohort  3

Three subjects received an IV infusion of SBC-102 at 3 mg · kg ⁻¹ . Dosing to the first subject in Cohort 3 and monitoring for tolerability for at least 24 hours prior to proceeding to the other two subjects in Cohort. Each subject remains an inpatient for 24 hours after the first injection of SBC-102. Subjects continued IV infusion once every 3 weeks for an additional 3 mg · kg ⁻¹ , provided that tolerability and safety remain acceptable.

 The Safety Committee (SC) may defer the dosing to the entire cohort or to individual subjects at any point due to poor tolerability or potential safety risks.

If the study is discontinued at a scheduled visit other than the eighth visit (termination of study), the subject should return seven days after the last dose of SBC-102 for the results of the study evaluation conducted at the eighth visit.

SBC-102 is administered by IV infusion on the second, fourth, fifth and sixth visits. Concurrent treatments are recorded throughout the study. Record side effects at the time of signature of the informed consent.

Each subject receives a total of four SBC-102 doses per week, provided that tolerability and safety remain acceptable.

Research Duration

The study involved a four-week dosing and washout period with SBC-102 to assist in the evaluation of safety and dosing schedules for subsequent clinical trials. After completion of this study, subjects can resume SBC-102 under separate protocols to assess long-term safety and efficacy of SBC-102 in patients with LAL deficiency / CESD phenotype.

Physical examination

Perform a general physical examination by a medically qualified person. The system (including but not limited to cardiovascular, respiratory, gastrointestinal and nervous systems) should be specified and documented. Any anomalies should be addressed at each time point and the test performed. New anomalies should be recorded as side effects, if applicable.

Evaluation of additional physical examinations performed in the screening method:

a) Liver size: Makes a clinical assessment of liver size (detectable / undetectable and centimeters below the leading edge), regularity (smooth / nodular) and sensitivity (stitching / not tingling).

b) Lymphadenitis : An assessment of the size, location and any detectable characteristic of any lymph node. Areas examined include the following: head (back head, front anterior, posterior ear, submandibular, submandibular gland), cervix, collarbone, armpits and inguinal area. Any extended node is characterized by stinging or not stinging.

c) Arterial disease: Clinically assess the right and left posterior tibial muscle and foot limb artery rhythm and record the right and left ankle brachial indexes (ABI). ABI is defined as the ratio of systolic blood pressure divided by right and left brachial systolic blood pressure (higher) in the limb artery or posterior tibial artery.

Vital signs

Vital signs, including pulse rate, respiratory rate, heart contraction, and diastolic blood pressure and body temperature (rectal or oral) are measured. Subjects are placed in semi-supine position for at least 5 minutes before assessing pulse rate and blood pressure. On the dosing day, vital signs are recorded before infusion, every 15 minutes (± 5) during the infusion and 2 hours after the infusion, then every 30 minutes (± 10) 2 to 4 hours after the infusion is complete. Additional readings may be taken at the investigator's discretion in the event of an infusion related reaction (IRR). The subject is placed supine for at least 5 minutes and then taken a 12-lead electrocardiogram (ECG) as a formal record.

Laboratory evaluation

Samples for laboratory testing are collected at the time indicated on the evaluation schedule. The following assays (except ESR, coagulation studies and anti-SBC-102 antibodies) are performed.

CBC / hematology: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean blood cell volume (MCV), mean blood cell hemoglobin (MCH), mean blood cell hemoglobin concentration (MCHC), platelet count, neutrophils, lymphocytes, monocytes, eosinophils, basophils

Chemical panel: glucose, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, magnesium, inorganic phosphorus, whole protein, lactate dehydrogenase, uric acid

Liver function test: AST / SGOT, ALT / SGPT, alkaline phosphatase, GGTP, albumin, bilirubin (direct, whole)

Lipid Panels: Total Cholesterol, Triglycerides, HDL, LDL

Coagulation Study: Prothrombin time (PT) international normalized ratio (INR), activated partial thromboplastin time (aPTT)

Urinalysis: glucose, ketones, blood, pH, protein, nitrate and white blood cells (microscopic examination may be done if blood, protein, nitrate and / or leukocytes are abnormal)

Viral infection screening: HBsAg and HCV serology (if clinically indicated at screening or during testing)

Autoimmune Hepatitis Screening: anti-smooth muscle antibody (ASMA), anti-nuclear antibodies (ANA), anti-LKM1 antibodies, anti-SLA antibodies

Anti-Drug Antibodies: Anti-SBC-102 Antibodies

Acute phase reactants: high sensitivity C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and serum ferritin

Pregnancy Test: All women have a pregnancy test at least every month. They are performed using serum for the first and eighth visits and urine for the second and sixth visits.

Pharmacokinetic ( PK ) Assessment: PK samples are taken from the arm opposite the infusion cannula. Intensive sampling for determination of SBC-102 serum levels was performed on Days 0 (dose 1 and 2) and Day 21 (doses 4 and 6); Immediately before dosing (within 30 minutes of dosing); At 10 (± 1), 15 (± 1), 20 (± 1), 40 (± 2), 60 (± 2) and 90 (± 2) minutes during and at the end of the infusion (approximately 120 minutes); And 5 (± 1), 10 (± 1), 20 (± 1), 30 (± 1), 40 (± 2), 60 (± 2) and 120 (± 2) minutes after completion of the infusion. .

SBC -102 manufacture

The body weight of the subject recorded in the first visit is used to calculate the SBC-102 volume for each infusion. SBC-102 drug product for IV infusion is prepared by dilution using the following steps:

1. Remove the vial from the refrigerator.

2. Check that the expiration date on the vial has not passed.

3. Determine the calculated total volume of SBC-102 required for dosing.

Yes:

Subject weight (in kg): 70 kg

Subject dose level: 3 mg kg ^-1

Drug concentration: 2.0 mg ml ^-1

One. Subject  Calculation of capacity:

2. Calculation of injection volume:

4. Use the following 0.9% saline infusion bags based on the medication group mission:

5. Remove the same volume of SBC-102 required for dosing (calculated in step 3 above) from a 100 ml or 250 ml infusion bag of 0.9% saline (ie, from the 250 ml infusion bag using the example above). 105 ml saline is removed).

6. Recover the calculated total volume of SBC-102 against the 0.9% saline infusion bag and transfer (ie, use the example above to recover 105 ml of SBC-102 solution and transfer to the infusion bag).

7. Carefully turn over to blend the bag.

rhLAL Dosing

1. Attach IV infusion tubing to the dilution bag of SBC-102.

2. Prepare the tubing and let all the air out.

3. Set the infusion rate in the flow-control device so that the entire volume is administered at approximately the following rate over approximately 100 minutes:

4. Select an IV infusion site that depends on the subject and may include the main vein or the wrist vein (or central venous catheter).

 5. The IV tubing is attached to the vascular catheter. Saline is injected into the IV line to assess efficacy and confirm that saline readily flushes.

6. Secure the IV wire with tape.

7. Start the SBC-102 injection using the flow-control device.

8. Monitor the infusion regularly.

9. When the bag is empty, 25 ml of 0.9% saline is injected directly into the infusion bag using the injection port.

10. Same injection rate until completion of infusion (60 ml per hour for 0.35 mg · kg ⁻¹ and ¹ mg · kg ⁻¹ doses [1 ml per minute] 150 ml per hour for ³ mg kg ⁻¹ doses 2.5 ml per minute]). The final injection is defined when the injection and flush are complete and verified.

Injection reaction

Infusion-related responses (IRRs) are defined as at least possibly any immunologically-mediated side effects associated with infusion. IRR is classified as acute (occurring within 24 hours of infusion) or delayed (occurring 1-14 days after infusion).

Agents and apparatus for the treatment of hypersensitivity reactions should be readily available in case of unexpected, severe hypersensitivity reactions. These feeds include, but are not limited to, oxygen, acetaminophen, antihistamines (eg, diphenhydramine, parenteral and PO), corticosteroids, epinephrine (adrenaline), and cardiopulmonary resuscitation devices.

Possible signs of acute IRR include redness, flushing, fever and / or chills, nausea, pruritus, urticaria, gastro-intestinal symptoms (vomiting, diarrhea, abdominal cramps), chest pain, shortness of breath, wheezing, wheezing, hypotension or high blood pressure It may be a cardiopulmonary reaction. If any of the above signs and symptoms are observed during infusion and the subject remains hemodynamically stable: the infusion rate should be slowed or moderated. If the subject continues to show signs of hypersensitivity, IM or a slow IV dose of antihistamine should be administered. In subjects who have experienced a severe infusion reaction with clinically significant cardiovascular or respiratory effects, the infusion is discontinued. In this hypersensitivity reaction, the subject may be treated with intravenous antihistamines, corticosteroids and epinephrine.

Example  8

Rat  Recombination within the model LAL Dosing

Repeated-dose effects by recombinant human LAL on body weight, tissue triglycerides and cholesterol, hepatomegaly, splenomegaly, lymphadenitis, intestinal weight and other variables are described in full herein by Yoshida and Kuriyama (1990) Laboratory Animal Science, vol 40, p 486-489 (also see Kuriyama et al (1990) Journal of Lipid Research, vol 31, p 1605-1611; Nakagawa et al, (1995) Journal of Lipid Research, vol 36, p 2212-2218), was evaluated in LAL deficient Donryu rats. At 4 weeks of age, pig rats homozygous for LAL deficiency (LAL − / −) were assigned to groups dosed with recombinant human LAL or saline placebo generated in a transgenic chicken fallopian system. Wild-type, age-matched, litter rats were used as controls. LAL-/-rats were tail-veined once a week for 4 weeks (4 doses total) or once every 2 weeks for 4 weeks (2 doses total) or as 2 doses given at 30 minute intervals Dosing by injection. The dose of recombinant LAL was 1 mg / kg or 5 mg / kg. The dosing schedule is shown in Table 5. To respond to potential hypersensitivity reactions, rats were pretreated with diphenhydramine (5 mg / kg) and with procedures based on previous experience in animal models of enzyme replacement therapy for the treatment of lysosomal reservoir (Shull et. (1994) Proceedings of the National Academy of Science, vol 91, p. 12937; Bielicki et al. (1999) The Journal of Biological Chemistry, 274, p.36335; Vogler et al. (1999) Pediatric Research, 45 , p.838.), the disclosures of which are incorporated herein by reference in their entirety.

FIG. 9 shows daily progression of weight gain in rats administered with 1 mg / kg recombinant LAL per week or 5 mg / kg recombinant LAL per week or 5 mg / kg recombinant LAL per week. It can be seen from the figure that there is little or no difference in treatment effect between the two dose sizes and frequency.

Example  9

Recombination LAL Treated with LAL  - / - Rat  Pathological examination

At the end of the study described in Example 8, the study animals were humanely euthanized and dissected to examine gross pathology, histopathology and clinical chemistry. Visual dissection included examination of the exterior of the body, all orifices and skulls, chest and abdominal cavity and its contents. Rats were weighed internal organs and tissues, organs and tissues were harvested and fixed in 10% neutral-buffered formalin. After fixation, tissues were processed and histological slides of the hematoxylin and eosin-staining sections were prepared and evaluated.

Gross pathology examination of the treated animals analyzed showed substantial normalization in liver size and color as seen in the anatomy shown in FIG. 10. The organ-to-weight ratio was determined and demonstrated a reduction in relative organ size for liver, spleen, mesenteric tissue, duodenum, jejunum, and ileum in successfully treated animals dissected compared to placebo treated rats. Histopathology of liver tissue from the recombinant LAL-treated rats analyzed shows hepatic cell pathology that is essentially normal, in marked contrast to the substantial accumulation of foam macrophages in placebo-treated animals (FIG. 10).

Example  10

Recombinant Humans in Macrophages and Fibroblast Lysosomes LAL Internalization of

The ability of transgenic algae-derived recombinant human LAL (“SBC-102”) to bind cells and internalize in lysosomal compartments was tested in vitro using macrophages and fibroblasts. When incubated with macrophage cells, it was found that fluorescently-labeled SBC-102 locates the lysosomes. This effect could be attenuated by using mannose polysaccharide competitors, indicating that N-acetylglucosamine / mannose (GlcNAc / mannose) receptors are involved as a mechanism of recognition and uptake by these cells. SBC-102 increased cell-bound LAL activity in both LAL-deficient human fibroblasts and normal murine fibroblasts after in vitro incubation, indicating that exposure to SBC-102 can result in substantial replacement of deficient enzyme activity. .

Mannose-6-phosphate (M6P) is present in the oligosaccharide structure of SBC-102 which has been shown to be involved in the delivery of lysosomal enzymes to a wide variety of cells via the ubiquitous M6P receptor.

Recombinant LALs were purified from the whites of transgenic hens. Oregon Green NHS was obtained from Invitrogen ™ (# 0-10241). Rat alveolar macrophage line NR8383 and mouse fibroblast line NIH-3T3 were obtained from ATCC. LAL-deficient Wolman fibroblasts were obtained from Coriell Institute for Medical Research and LysoTracker® Red was obtained from Invitrogen®.

Enzyme Labeling : 4 mg of transgenic algal derived LAL in PBS was labeled with Oregon Green according to the manufacturer's instructions, after which the reaction was dialyzed against PBS and then concentrated.

Macrophage uptake : Fluorescently-labeled transgenic algae derived LAL (5 μg / ml) and LysoTracker® Red were incubated with NR8383 cells for 2 hours. Cells were examined by confocal fluorescence microscopy using a continuous scanning scheme at 488 nm and then 514 nm.

Competitive Inhibition by Mannan: Fluorescently-labeled SBC-102 (5 μg / ml) and mannan were incubated with NR8383 cells for 2 hours. Cells were trypsinized and recombinant LAL uptake was measured by fluorescence-activated cell sorting using median fluorescence intensity as endpoint.

The ability of the transgenic LALs to be nested within the lysosomes of the target cells and subsequently included was tested using the macrophage cell line NR8383. Fluorescently-labeled transgenic algae derived LAL and lysosomal marker "LysoTracker® Red" (Invitrogen®) were incubated with the cells for 2 hours. Colocalization of transgenic algae-derived LAL and lysosomal markers in the lysosomes of these cells was then examined by confocal fluorescence microscopy using a sequential scanning approach (FIG. 11). Recombinant LAL demonstrated localization to lysosomes, consistent with similar in vitro studies using recombinant humans (rhLAL) from various sources.

The binding specificity of transgenic algae-derived LALs to GlcNAc / mannose receptors was assessed by competitive binding assays using the macrophage cell line NR8383 (FIG. 12). Fluorescent-labeled (Oregon Green) transgenic algal-derived LALs and various concentrations of mannose-containing oligosaccharides, ie, manna, were co-incubated with the cells for 2 hours at 5 μg / ml. Relative inhibition of transgenic algae-derived LAL uptake by mannan was compared by fluorescence-activated cell sorting assay using median fluorescence intensity as the endpoint as compared to the control without mannan. Mannose dose dependent inhibition was observed in transgenic algal derived LAL binding / absorption, consistent with the transgenic algal derived LAL: GlcNAcR interaction.

In addition, mannose-6-phosphate mediated uptake in fibroblasts was demonstrated by competition experiments with mannose-6-phosphate.

Example  11

Intracellular treated LAL  Increase in activity

LAL catalyzes the hydrolysis of cholesterol esters and triglycerides into free cholesterol, glycerol and free fatty acids. Thus, LAL activity can be measured, for example, by cleavage of the fluorescent substrate, 4-methylumbeliferyl oleate (4MUO).

Fibroblast

The ability of transgenic algal-derived LAL exposure to increase intracellular LAL activity was tested using normal cells and LAL-deficient cells in vitro. Fibroblasts were isolated from menopausal patients and normal murine fibroblasts (NIH-3T3) were incubated in the presence of transgenic algae derived LAL for 5 hours at concentrations of 0, 0.16 or 0.5 micrograms / ml. Cells were then washed to remove non-specific signals, and cell lysates were analyzed using 4-methylumbeliferyl oleate (4-MUO) substrate. FIG. 13 demonstrates that endogenous cell-binding LAL activity is lower in fibroblasts compared to NIH-3T3, and a dose-dependent increase in LAL activity was observed in both cell types after incubation with transgenic algal-derived LAL. (FIG. 13).

leukocyte

Serum protein leukocytes were obtained before and after dosing from LAL deficient patients. Blood samples were stored refrigerated without compromising enzyme activity. Mononuclear leukocytes (lymphocytes) were isolated from blood using the preparation of Ficoll and sodium diatrazoate. 4-8 mL blood, previously diluted 1: 1 with Hanks' balanced salt solution, was gently layered and centrifuged over 3 mL Ficoll-Paque. Monocyte rings were aspirated and washed at least twice by washing with Hanks' solution and then resuspending the pellet in 1-2 ml water. Pellets were frozen at −20 ° C. prior to use. Prior to analysis, the pellets were thawed, resuspended in distilled water and sonicated on ice. The preparation was then centrifuged at 20,000 × g for 15 minutes at 4 ° C. Supernatants (containing 0.5-1.5 mg protein / ml) were kept on ice prior to analysis.

By the addition of 10 mM 1㎖ 4MUO (4- methyl Titanium Valley perylene oleate) in hexane in CHCI ₃ in 16 mM L-α- phosphatidylcholine 1㎖ to prepare a substrate for an acid lipase analysis. The solvent was evaporated under N ₂ and 25 ml 2.4 mM taurodeoxycholic acid (sodium salt) in water was added. The mixture was sonicated on ice for 1-2 minutes at 30-40 W. Prior to analysis, one volume of substrate stock was diluted with seven volumes of 200 mM sodium acetate / acetic acid buffer, pH 4.0. Each 2 ml reaction cuvette contained 100 mmol 4-methylumbelliferyl oleate, 160 mmol L-α-phosphatidylcholine and 600 mmol sodium taurodeoxycholate.

The reaction was started by adding 5-100 μl enzyme and monitored at 37 ° C. using a spectrophotometer. Cleavage of 4MUO was detected by excitation of the released fluorophore, 4-methylumbelliferone (4MU), for example at about 360 nm and emission at about 460 nm. The change in fluorescence over time was recorded.

Example  12

In Vivo Analysis of Recombinant Human LAL (SBC-102)

LAL-deficient Yoshida rats (ie, isoforms) (Kuriyama et al. (1990), Journal of Lipid Research, vol. 31, p 1605-1611; Nakagawa et al., (1995) Journal of Lipid Research , vol. 36, p 2212-2218; and Yoshida and Kuriyama (1990) Laboratory Animal Science, vol. 40, p 486-489), starting at 4 weeks of age, and once weekly for 4 weeks SBC-102 ( 5 mg / kg, IV) or placebo. For each administration, SBC-102 was injected into the tail vein twice at the same dose (2.5 mg / kg) 30 minutes apart. Rats and age-matched wild-type controls were tested 1 week after the last dose. The analysis was performed three times.

Gross pathology examination of SBC-102 treated animals demonstrated normalization of liver color in addition to a reduction in organ size. SBC-102 treated rats exhibited essentially normal liver tissue in marked contrast to the substantial accumulation of foam macrophages in vehicle treated animals. Elevated serum alanine and asphaltate transferase levels in LAL ^{− / −} rats were also reduced in SBC-102 treated rats.

Mass of internal organs and tissues was determined for each rat and data is shown in FIG. 14. Organ size is expressed as a percentage of body weight determined at 8 weeks of age in LAL ^{− / −} and LAL ^{+ / +} rats after 5 mg / kg weekly administration of vehicle or SBC-102 for 4 weeks.

As shown in FIG. 15, the weights of SBC-102- or vehicle-treated Yoshida rats were compared to wild type rats. SBC-102 (5 mg / kg) or vehicle was administered by IV injection to LAL ^{− / −} rats as single dose or as divided doses (given within a 4 hour period). LAL ^{+ / +} rats were age-matched litter controls.

Example  13

Triglycerides  analysis

Triglyceride assays were performed on liver and spleen tissue from wild-type placebo and isoform SBC-102 treated animals. Triglyceride analysis was performed using standard methods (ie, triglyceride quantification kit catalog # JM-K622-100 from MBL International).

Wild type and LAL  lack of In the rat  Liver and spleen Triglycerides  level  Triglyceride (μg / mg wet tissue) Wild type (n = 3) Placebo (n = 3) SBC-102 (n = 3) liver 48 84 57 spleen 3 22 4

Liver stroma levels

FIG. 16 shows cholesterol, cholesteryl ester and triglyceride levels determined at 8 weeks of age in WT and LAL deficient rats after 5 mg / kg ⁻¹ weekly administration of vehicle or SBC-102 for 4 weeks.

Example  14

Rat  Dose Response Study

Based on the studies performed above, the pharmacodynamic (PD) effects of dose and dose schedule range of LAL (“SBC-102”) were tested in LAL ^{− / −} rats. In these studies, SBC-102 was administered at 4 weeks of age starting at weekly doses of 0.2, 1, 3 and 5 mg / kg for one month or weekly (qw) for 0.35, 1.0 and 5.0 mg / kg. Administration was by IV injection. The results show improvement in body weight (BW) gain (FIG. 17), hospital prescription (FIG. 18) and tissue substrate levels (FIG. 19). Serum transaminase levels also decreased with increasing SBC-102 doses, and levels reached essentially wild-type levels at higher doses.

Example  15

SBC Pharmacokinetics of -102

a. sampling

PK samples were taken from adult patients with late LAL deficiency. Patients were dosed at 0.35 mg / kg for 2 hours. Serum samples from Days 0 (dose 1 and 2) and Day 21 (doses 4 and 6) were immediately prior to dosing (within 30 minutes of dosing); During the infusion (DI) and at the end of the infusion (EOI) (approximately 120 minutes from the beginning of the infusion) 10 (± 1), 15 (± 1), 20 (± 1), 40 ( ± 2), 60 (± 2) and 90 (± 2) minutes; And 5 (± 1), 10 (± 1), 20 (± 1), 30 (± 1), 40 (± 2), 60 (± 2) and 120 (± 2) minutes after completion of the infusion. .

b. Serum Enzyme Analysis

4-MUO (4 mM) kept in a -20 ° C. freezer was thawed in a 4 ° C. refrigerator in the dark and placed in a 25 ° C. incubator for 1.5 hours in the dark before use. Standards were prepared by diluting the SBC-102 drug product to 1.56 ng / ml. Blank assay buffer was included. All samples were diluted to 50 ng / ml for the first dilution. Standards and samples were plated immediately after the dilution was made. After preparing the standards and samples, 62.5 μl of assay buffer (0.2 mol / L sodium acetate trihydrate, pH 5.5) was added to each well. 12.5 μl of standard and sample were added twice to each well. 4-MUO (4 mM) was diluted to 1.6 × with 4% Triton X-100 and 25 μl per well was added. The multi-well plate is tapped several times to mix, tightly sealed and placed in a 37 ° C. incubator for 30 minutes. After incubation, 50 μl of stop solution (0.77M Tris pH 8.0) is added to each well to make a final volume of 150 μl / well. The plate was placed on a microplate reader and the fluorescence level was measured from the bottom of the plate at excitation at 360 nm and emission at 460 nm.

As shown in Tables 7-11, the serum C _max of recombinant LAL administered to adult patients with late LAL deficiency ranges from approximately 270 ng / ml to 720 ng / ml. Half-life (t _1/2) is in the range of 7.6 minutes to 16.7 minutes and an average t _1/2 was approximately 13 minutes (standard deviation 3.812).

patient ID  (Capacity: 0.35 mg Of kg ) visit # density( ng / Ml) Nominal time 02-001 2 5.63 Before injection 02-001 2 241.88 10 minutes DI 02-001 2 369.44 15 minutes DI 02-001 2 369.93 20 minutes DI 02-001 2 359.64 40 minutes DI 02-001 2 294.64 60 minutes DI 02-001 2 71.85 90 minutes DI 02-001 2 71.20 EOI 02-001 2 37.95 5 minutes AI 02-001 2 24.91 10 minutes AI 02-001 2 14.16 20 minutes AI 02-001 2 9.76 30 minutes AI 02-001 2 9.02 40 minutes AI 02-001 2 7.20 60 minutes AI 02-001 2 7.51 120 minutes AI C _max = 369.93 ng / ml; t _1/2 = 16.8 min; LLOQ = 4.68 ng / ml

patient ID  (Capacity: 0.35 mg Of kg ) visit # density( ng / Ml) Nominal time 03-001 2 8.23 Pre 03-001 2 199.80 10 minutes DI 03-001 2 215.10 15 minutes DI 03-001 2 228.12 20 minutes DI 03-001 2 237.25 40 minutes DI 03-001 2 210.73 60 minutes DI 03-001 2 262.41 90 minutes DI 03-001 2 102.39 EOI 03-001 2 52.20 5 minutes AI 03-001 2 33.75 10 minutes AI 03-001 2 17.60 20 minutes AI 03-001 2 12.17 30 minutes AI 03-001 2 10.82 40 minutes AI 03-001 2 9.39 60 minutes AI 03-001 2 8.72 120 minutes AI C _max = 262 ng / ml; t _1/2 = 15.3 min; LLOQ = 4.68 ng / ml

patient ID  (Capacity: 0.35 mg Of kg ) visit # density( ng / Ml) Nominal time 03-002 2 <4.68 Before injection 03-002 2 480.55 10 minutes DI 03-002 2 531.16 15 minutes DI 03-002 2 613.85 20 minutes DI 03-002 2 717.75 40 minutes DI 03-002 2 84.46 60 minutes DI 03-002 2 54.34 90 minutes DI 03-002 2 171.41 EOI 03-002 2 87.95 5 minutes AI 03-002 2 49.70 10 minutes AI 03-002 2 16.47 20 minutes AI 03-002 2 11.01 30 minutes AI 03-002 2 9.76 40 minutes AI 03-002 2 6.28 60 minutes AI 03-002 2 5.19 120 minutes AI C _max = 718 ng / ml; t _1/2 = 10.8 min; LLOQ = 4.68 ng / ml

patient ID  (Capacity: 0.35 mg Of kg ) visit # density( ng / Ml) Nominal time 02-001 6 5.12 Before injection 02-001 6 249.91 10 minutes DI 02-001 6 294.37 15 minutes DI 02-001 6 313.75 20 minutes DI 02-001 6 330.04 40 minutes DI 02-001 6 245.48 60 minutes DI 02-001 6 262.78 90 minutes DI 02-001 6 81.09 EOI 02-001 6 32.10 5 minutes AI 02-001 6 20.39 10 minutes AI 02-001 6 10.74 20 minutes AI 02-001 6 8.08 30 minutes AI 02-001 6 6.38 40 minutes AI 02-001 6 5.82 60 minutes AI 02-001 6 <4.68 120 minutes AI C _max = 330 ng / ml; t _1/2 = 15.3 min; LLOQ = 4.68 ng / ml

patient ID  (Capacity: 0.35 mg Of kg ) visit # density( ng / Ml) Nominal time 03-001 6 6.63 Before injection 03-001 6 317.45 10 minutes DI 03-001 6 333.46 15 minutes DI 03-001 6 310.83 20 minutes DI 03-001 6 378.68 40 minutes DI 03-001 6 234.49 60 minutes DI 03-001 6 246.07 90 minutes DI 03-001 6 206.06 EOI 03-001 6 253.80 5 minutes AI 03-001 6 70.81 10 minutes AI 03-001 6 24.61 20 minutes AI 03-001 6 12.32 30 minutes AI 03-001 6 9.03 40 minutes AI 03-001 6 7.18 60 minutes AI 03-001 6 5.56 120 minutes AI C _max = 379 ng / ml; t _1/2 = 7.7 min; LLOQ = 4.68 ng / ml

Example  16

Immunogenicity Analysis: Anti- SBC Measurement of -102

Each unknown positive and negative sample was diluted 1:20 in 5% milk powder in 1 × PBS and incubated on rotator (500 rpm) for 12-18 hours at 4 ° C. Prior to analysis, samples were centrifuged at 2000 × g for 20 minutes and the supernatants were removed with fresh 1.5 ml tubes.

SBC-102 was diluted to 0.5 μg / ml in IX PBS buffer and 100 μl was placed in each well of a 96-well ELISA plate. The plate was covered with an adhesive cover and incubated for 8 hours at room temperature or overnight at 4 ° C. After incubation, the wells were washed three times with 1 × wash buffer. 200 μl of 5% BSA-IgG glass was added to each well, the plate was sealed and incubated at 4 ° C. for 12-18 hours or at room temperature for 2 hours. After incubation, the wells were washed three times with 1 × wash buffer. 100 μl of each control sample, unknown sample and negative sample were added to the wells three times. Plates were incubated on a microplate shaker (500 rpm) for 1.5 hours at room temperature. After incubation, the wells were washed three times with 1 × wash buffer. 100 μl of biotinylated SBC-102 diluted in dilution buffer at a concentration of 100 ng / ml was added to each well, and the plates were incubated on a microplate shaker (500 rpm) for 1.5 hours at room temperature. After incubation, wells were washed with 1 × wash buffer. 100 μl of streptavidin-HRP conjugate diluted 1: 4000 in dilution buffer was added to each well. Plates were incubated at room temperature for 1.5 hours on a microplate shaker (500 rpm). After incubation, the wells were washed four times with 1 × wash buffer. 100 μl of TMB substrate was added to each well and the plate was incubated for 15 minutes in the dark. 50 μl of stop solution (0.5NH ₂ SO ₄ ) was added to each well to stop the reaction. OD was measured at 450 nm.

As shown in Table 12, patients receiving a dose of 0.35 mg / kg SBC-102 weekly for 4 weeks did not show elevated levels of anti-SBC-102 antibody, which indicated that enzyme replacement treatment by SBC-102 infusion Does not cause any significant immunogenicity in human patients. These patients did not show any side effects or infusion-related responses (IRRs).

Example  17

Recombination LAL By administration of Of moonshine (WL)  cure

The female patient was hospitalized at 7 weeks of age due to the difficulty of weight gain after birth and poor progress. At the first physical examination, the patient weighed 3.6 kg (birth weight 3.7 kg), was dry and the skin wrinkles were loose. The abdomen is swollen and there is a clear hepatomegaly of 6 cm and a clear spleen of 4 cm. Note the enlarged lymph nodes in the inguinal area, weak muscle activity.

Initial hemoglobin levels are 9.2 gm, platelets are 506,000 and leukocytes are 11,550. Urinalysis is normal and bone marrow smears show vacuole lymphocytes and numerous foam cells. Serum chemistry measurement: total lipid 834 mg / 100 ml, phospholipid 176 mg / 100 ml, triglyceride 141 mg / 100 ml, cholesterol 129 mg / 100 ml, bilirubin 0.3 mg / 100 ml, alkaline phosphatase 9.0 BU%, 90 units of SGOT, 50 units of SGPT, 20 units of cholinesterase, 8.3 mg of urea nitrogen, 45 mg / 100 ml per fasting. CT scan of the abdomen shows adrenal gland enlarged bilaterally with hepatomegaly and calcification.

The patient is surgically implanted with a venous vascular access port for administration. After connecting ports to mobile injectors, patients are pretreated with 1 mg / kg of diphenhydramine 20 minutes prior to recombinant LAL infusion to counteract potential hypersensitivity infusion reactions. The patient is then administered recombinant LAL at 1 mg / kg over a 5 hour course by intravenous infusion. This treatment is repeated indefinitely once every 7 days.

Within two weeks of administration of the first dose of recombinant LAL, the patient is assessed for weight gain and size of significant abdominal organs as determined by ultrasound. Laboratory results of testing lysosomal acid lipase activity in patients are also performed.

Example  18

Recombination LAL By administration of Cholesteryl  Ester storage diseases ( CESD Treatment of

A 3-year-old boy with a pruritus abdominal rash is examined by his pediatrician. During the abdominal examination, hepatomegaly is noticed by the doctor and confirmed by ultrasound. Patients are regularly monitored at this undiagnosed point.

He is hospitalized at age 8 with gastroenteritis. Light microscopy of liver biopsies reveals glycogen and small lipid droplets in the cytoplasm of hepatocytes. Electron microscopy shows droplets of membrane-bound lipids with small electron density granules. Occupational diagnosis of glycogen storage disease type III (Debrancher disease) is made, but dermal fibroblast debrancher activity is normal.

Hepatomegaly continues at age 10, with a second liver biopsy. Light microscopy shows altered lobular structure of the hepatic dermal tissue with expanded hepatocytes containing intracellular granules and vacuoles with mild periportal fibrosis. Fibroblast acid lipase activity is found to be 7% of normal and confirms the diagnosis of CESD. Plasma concentrations of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were found at the 95th percentile for age and gender at 7.51, 3.24 and 5.58 mmol / L, respectively. Whereas, plasma high-density lipoprotein cholesterol (HDL-C) is below the lower fifth percentile at 0.47 mmol / L; He has combined hyperlipidemia (hypercholesterolemia, hypertriglyceridemia and hypoalphalipoproteinemia).

The patient is surgically implanted with a venous vascular access port for administration. After connecting the port to the mobile injector, the patient is pretreated with 5 mg / kg of diphenhydramine before 20 recombinant LAL infusions to counter a potential hypersensitivity infusion reaction. The patient is then administered recombinant LAL at 5 mg / kg over a 5 hour course by intravenous infusion. This treatment was repeated indefinitely once every 14 days.

Within two weeks of administration of the first dose of recombinant LAL, the patient is assessed for weight gain and size of the abdominal organs as determined by ultrasound. Laboratory results of testing lysosomal acid lipase activity in patients are also performed.

Each embodiment of the specification is provided by way of explanation of the invention, but is not a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and variations can be made without departing from the scope or spirit of the invention. For example, features described or described as part of one embodiment may be used in another embodiment to still obtain further embodiments. The present invention is intended to address these variations, combinations, additions, deletions and variations.

All publications, patents, patent applications, Internet sites, and accession numbers / database sequences (polynucleotide and polypeptide sequences) cited herein are referenced by their respective publications, patents, patent applications, Internet sites, and accession numbers / database sequences. It is hereby incorporated by reference in its entirety for all purposes to the same extent as specifically and individually indicated to be incorporated by reference.

                         SEQUENCE LISTING <110> SYNAGEVA BIOPHARMA CORP.   <120> USE OF LYSOSOMAL ACID LIPASE FOR TREATING LYSOSOMAL ACID LIPASE DEFICIENCY IN PATIENTS <130> 121424-00420 <140> PCT / US2011 / 051096 <141> 2011-09-09 <150> 61 / 403,011 <151> 2010-09-09 <150> 61 / 456,014 <151> 2010-10-29 <150> 61 / 432,372 <151> 2011-01-13 <150> PCT / US2011 / 033699 <151> 2011-04-23 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 378 <212> PRT <213> Homo sapiens <220> <223> LAL <400> 1 Ser Gly Gly Lys Leu Thr Ala Val Asp Pro Glu Thr Asn Met Asn Val 1 5 10 15 Ser Glu Ile Ile Ser Tyr Trp Gly Phe Pro Ser Glu Glu Tyr Leu Val             20 25 30 Glu Thr Glu Asp Gly Tyr Ile Leu Cys Leu Asn Arg Ile Pro His Gly         35 40 45 Arg Lys Asn His Ser Asp Lys Gly Pro Lys Pro Val Val Phe Leu Gln     50 55 60 His Gly Leu Leu Ala Asp Ser Ser Asn Trp Val Thr Asn Leu Ala Asn 65 70 75 80 Ser Ser Leu Gly Phe Ile Leu Ala Asp Ala Gly Phe Asp Val Trp Met                 85 90 95 Gly Asn Ser Arg Gly Asn Thr Trp Ser Arg Lys His Lys Thr Leu Ser             100 105 110 Val Ser Gln Asp Glu Phe Trp Ala Phe Ser Tyr Asp Glu Met Ala Lys         115 120 125 Tyr Asp Leu Pro Ala Ser Ile Asn Phe Ile Leu Asn Lys Thr Gly Gln     130 135 140 Glu Gln Val Tyr Tyr Val Gly His Ser Gln Gly Thr Thr Ile Gly Phe 145 150 155 160 Ile Ala Phe Ser Gln Ile Pro Glu Leu Ala Lys Arg Ile Lys Met Phe                 165 170 175 Phe Ala Leu Gly Pro Val Ala Ser Val Ala Phe Cys Thr Ser Pro Met             180 185 190 Ala Lys Leu Gly Arg Leu Pro Asp His Leu Ile Lys Asp Leu Phe Gly         195 200 205 Asp Lys Glu Phe Leu Pro Gln Ser Ala Phe Leu Lys Trp Leu Gly Thr     210 215 220 His Val Cys Thr His Val Ile Leu Lys Glu Leu Cys Gly Asn Leu Cys 225 230 235 240 Phe Leu Leu Cys Gly Phe Asn Glu Arg Asn Leu Asn Met Ser Arg Val                 245 250 255 Asp Val Tyr Thr Thr His Ser Pro Ala Gly Thr Ser Val Gln Asn Met             260 265 270 Leu His Trp Ser Gln Ala Val Lys Phe Gln Lys Phe Gln Ala Phe Asp         275 280 285 Trp Gly Ser Ser Ala Lys Asn Tyr Phe His Tyr Asn Gln Ser Tyr Pro     290 295 300 Pro Thr Tyr Asn Val Lys Asp Met Leu Val Pro Thr Ala Val Trp Ser 305 310 315 320 Gly Gly His Asp Trp Leu Ala Asp Val Tyr Asp Val Asn Ile Leu Leu                 325 330 335 Thr Gln Ile Thr Asn Leu Val Phe His Glu Ser Ile Pro Glu Trp Glu             340 345 350 His Leu Asp Phe Ile Trp Gly Leu Asp Ala Pro Trp Arg Leu Tyr Asn         355 360 365 Lys Ile Ile Asn Leu Met Arg Lys Tyr Gln     370 375 <210> 2 <211> 21 <212> PRT <213> Homo sapiens <220> <223> Native signal peptide <400> 2 Met Lys Met Arg Phe Leu Gly Leu Val Val Cys Leu Val Leu Trp Thr 1 5 10 15 Leu His Ser Glu Gly             20

Claims (65)

A method of treating a human patient with lysosomal acid lipase (LAL) deficiency, comprising administering a recombinant human LAL to the patient, wherein said administration is sufficient to improve liver function. The method of claim 1, wherein the administration is sufficient to normalize the liver test. The method of claim 1 or 2, wherein the administration minimizes hepatomegaly. The method of claim 1, wherein the administration is sufficient to reduce liver size. 5. The method of claim 1, wherein the administration is sufficient to reduce serum levels of hepatic transaminase. 6. The method of claim 1, wherein the administration is sufficient to reduce lipid levels. The method of claim 1, wherein the administration is sufficient to reduce triglyceride levels. The method of claim 1, wherein the administration is sufficient to reduce the cholesteryl ester.  A method of increasing LAL activity in a human patient with LAL deficiency, comprising administering a recombinant human LAL to the patient, wherein said administering increases LAL activity in the patient. The method of claim 9, wherein the administration is sufficient to increase LAL activity by at least about 35 mmol / mg / min in the patient. A method of treating a disease associated with LAL deficiency in a patient in need thereof, comprising administering to said patient an effective amount of an exogenous LAL protein once every 5 to 30 days. A method for treating a disease associated with LAL deficiency in a patient in need of treatment for a disease associated with LAL deficiency, wherein the patient is administered a dose of exogenous LAL from 0.1 mg to 50 mg per kilogram once every 7 to 14 days. Method comprising the steps of: A method of treating a disease associated with LAL deficiency in a patient in need thereof, comprising administering to said patient a dose of exogenous LAL from about 0.1 mg to 5.0 mg per kilogram. A method of treating moonshine disease or CESD in a human patient in need of treatment of Wolman Disease or CESD, wherein the patient has a dose of about 0.1 mg to 50 mg of exogenous LAL per kilogram once every 30 days. Administering once every time. The method of claim 11, wherein the exogenous LAL is a recombinant human LAL. 16. The method of any one of claims 11-15, wherein the exogenous LAL comprises at least one mannose or mannose-6-phosphate. 17. The method of any one of claims 1-12 and 14-16, wherein the LAL is administered in an amount of about 0.10 to about 5.0 mg / kg. The method of claim 17, wherein the amount is about 0.30 mg / kg. The method of claim 17, wherein said amount is about 0.35 mg / kg. The method of claim 17, wherein the amount is about 1.0 mg / kg. The method of claim 17, wherein said amount is about 3.0 mg / kg. The method of any one of claims 1-21, which is administered almost weekly. The method of any one of claims 1-21, which is administered almost every two weeks. The method of claim 1, wherein the administration is sufficient to reduce the level of serum aspartate transaminase (AST) by at least 50% from the pretreatment level. The method of any one of claims 1-24, wherein the administration is sufficient to reduce the level of serum alanine transaminase (ALT) by at least 50% from the pretreatment level. To claim 1, wherein A method according to any one of claim 25, wherein the dosage will be sufficient to achieve a LAL half-life (t _1/2) of less than about 17 minutes. Any one of claims 1 to 26. A method according to any one of the preceding, the dosage is from about 6, 7, 8, 9, 10, 11, 12, 13, 14, (t 1/2) 15, 16 or LAL half-life of 17 minutes of anti- Sufficient to achieve that. The method of claim 1, wherein the administration is sufficient to achieve a C _max of at least about 200 ng per ml of serum. The method of any one of claims 1-28, wherein the administration is sufficient to achieve about 200 ng per ml of serum to 800 ng C _max per ml of serum. The method of any one of claims 1-29, wherein the LAL is internalized by fibroblasts. 31. The method of any one of claims 1-30, wherein the LAL is internalized by macrophages. 32. The method of any one of the preceding claims, wherein the LAL is produced by a transgenic bird. 32. The method of any one of the preceding claims, wherein the LAL is produced by a mammalian cell line. The method of claim 33, wherein the mammalian cell line is a human cell line. 35. The method of any one of claims 1 to 34, wherein the administration is intravenous administration. 36. The method of claim 35, wherein the administration is by infusion. The method of claim 36, wherein the infusion is about 2 hours to about 4 hours. 38. The method of claim 36 or 37, wherein the infusion is at a rate of about 0.1 mg / kg / hour to about 4 mg / kg / hour. The method of claim 1, wherein the administering minimizes the immune response to recombinant human LAL. 40. The method of any one of claims 1-39, wherein the administration reduces lymphadenitis. 41. The method of any one of claims 1-40, wherein the administration is sufficient to reduce serum ferritin levels. 42. The method of any one of claims 1-41, wherein the administration is sufficient to increase serum hemoglobin levels. 43. The method of any one of claims 1-42, wherein the administration is sufficient to increase growth in a child patient. 44. The method of any one of claims 1-43, wherein the administration is sufficient to increase the rate of weight gain. 45. The method of any one of claims 1-44, wherein the rate of growth of the patient after administration is at least about twice the rate of growth of the patient prior to administration. 46. The method of any one of claims 1-45, wherein administration is sufficient to increase adipose tissue. 47. The method of any one of claims 1 to 46, wherein the patient is less than 1 year old. 47. The method of any one of claims 1 to 46, wherein the patient is at least one year old. 49. The method of any one of claims 1-48, wherein the patient has been diagnosed with moonshine. 50. The method of any one of claims 1-49, wherein the patient has been diagnosed with Cholesteryl Ester Storage Disease (CESD). 51. The method of claim 50, wherein the CESD was diagnosed before age 10. The method of any one of claims 1-51, further comprising administering a second therapeutic agent. The method of claim 52, wherein the second therapeutic agent is a cholesterol-lowering drug. The method of claim 53, wherein the second therapeutic agent is statin. The method of claim 53, wherein the second therapeutic agent is ezetimibe. The method of claim 52, wherein the second therapeutic agent reduces the immunogenicity of the LAL. The method of claim 52, wherein the second therapeutic agent is antihistamine. The method of claim 52, wherein the second therapeutic agent is an immunosuppressive agent. 58. The method of claim 57, wherein the antihistamine is a pharmaceutically effective dose of diphenhydramine administered before LAL is administered. 60. The method of claim 59, wherein the dose of diphenhydramine is about 1 to about 5 mg per kilogram. 61. The method of claim 59 or 60, wherein diphenhydramine is administered about 20 to about 90 minutes prior to administration of LAL. 62. The method of any one of claims 1-61, further comprising completing an assessment of monitoring clinical and pathological features in the patient. 63. The method according to claim 62, wherein the evaluation is lipid analysis, chest x-ray, liver function test, stool chart, plasma mevalonic acid assay, immunogenicity test, plasma lysosomal acid lipase assay, chitotriocidase assay, portal hypertension assay, human body Metrology, liver or spleen characterization, gastrointestinal tract imaging, or a combination thereof. The method of claim 63, wherein the imaging technique is ultrasound, magnetic resonance imaging, or nuclear magnetic resonance spectroscopy. 65. A composition for administering LAL according to the method of any one of claims 1-64.

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