Using acid lysosome lipase for treating acid lysosome lipase deficiency in patients

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for treating a human patient suffering acid lysosome lipase (ALL) deficiency. That is ensured by administering human recombinant ALL into the above human patient in an amount effective to normalise hepatic transaminase in serum or blood. The above human recombinant ALL is administered once approximately every 7 days to once approximately every 30 days, and the above dose is adequate to reducing the hepatic involvement in the above human patient.

EFFECT: invention provides treating the ALL deficiency, such as Wolman disease, cholesterol ester storage disease (CESD) both at the early, and late stages of the disease.

32 cl, 19 dwg, 12 tbl, 18 ex

 

The LEVEL of TECHNOLOGY

Deficiency of acid lysosomal lipase (CFL) is a rare lysosomal storage disease, storage disease (LSD), characterized by the absence of cleavage of cholesterol ester (CE) and triglycerides (TG) in the lysosomes due to a deficiency of the enzyme. The shortage of CFLs has similarities with other lysosomal diseases of accumulation with accumulation of the substrate in several types of tissues and cells. With a deficit of CFLs accumulation of substrate is most noticeable in the cells of the reticuloendothelial system, including Kupffer cells in the liver and histiocytes in the spleen and in the lamina propria of the mucosa of the small intestine. Reticuloendothelial cells expressing the receptor of glucosamine mannose/N-acetyl macrophage (also known as the mannose receptor of macrophages, RMP or CD206), which stimulates the binding, the inclusion of cell substances and lysosomal internalization of proteins with N-acetylglucosamine or terminated N-glycans mannose and provides a way for a possible correction of the enzyme deficiency in these key types of cells.

The deficit CFL is a Multisystem disease that is most often manifested in gastrointestinal, hepatic and cardiovascular complications and is associated with significant morbidity and mortality. Clinical consequences of deficiency CFL arise as a result of observationally lipid material in the lysosomes in a number of tissues and drastic violations in the mechanisms of regulation of cholesterol and lipid histiocytosis, including a significant increase in hepatic synthesis of cholesterol. The deficit CFL is at least two phenotypes: Wolman disease (WD) and the disease is the accumulation of cholesterol esters (BNAH).

The Wolman disease, named after the doctor who first described it, is the most aggressive manifestation of deficiency of CFLs. This phenotype is characterized by gastrointestinal and hepatic manifestations, including impaired growth, malabsorbtsija, steatorrhea, significant weight loss, lymphadenopathy, splenomegaly and hepatomegaly. The Wolman disease progresses rapidly and invariably leads to death usually in the first year of life. Analysis of case histories shows that survival after 12 months of age is exceedingly rare for patients admitted with impaired growth due to severe shortage of CFLs in the first year of life. With this most aggressive form, the disturbance of growth is the dominant clinical feature and is a major contributor to mortality at an early age. Liver damage, manifested in the form of its increase and improve the level of transaminases, also is a common manifestation in infants.

The diagnosis of Wolman disease is based on physical indicators and the results of laboratory tests. Infants are usually hospitalized during the first two months�and life due to diarrhoea, persistent vomiting, difficulty in feeding, retarded growth, lack of development. Physical indicators include bloating of the abdomen due to hepatomegaly and splenomegaly, and radiographic examination often reveals calcification of the adrenal glands. Laboratory studies usually reveal elevated levels of serum transaminase levels and the absence or significant reduction in the activity of endogenous enzyme CFL. Some patients have elevated levels of cholesterol and triglycerides in the blood.

Patients with deficiency of CFLs also can be detected at a greater age mainly on the liver and cardiovascular system, and this state is often called a disease of accumulation of cholesterol esters (BNAH). When BIH, the liver is severely affected due to hepatomegaly, necrosis of liver cells, increased transaminase levels, cirrhosis and liver fibrosis. Due to the increased levels of CE and TG defeat of the cardiovascular system can be characterized by hyperlipidemia. Accumulation of fatty deposits on artery walls (atherosclerosis) was found in some patients suffering from BIH. The deposits narrow the arterial lumen and can lead to blockage of blood vessels, increasing the risk of cardiovascular complications, including myocardial infarction and sudden attacks. However, not all patients, stradus�x deficiency CFL, atherosclerosis develops. For example, in patients with Wolman disease have other symptoms associated with the disease, including an enlarged liver and spleen, lymphadenopathy and malabsorption of the small intestine, but BV in General not characterized by the occurrence of atherosclerosis (The Metabolic and Molecular Bases of Inherited Disease (the Metabolic and molecular basis of inherited diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle D., etc.) 7th edition. Volume 2, p. 2570, McGraw-Hill, 1995). Also not all patients with BIH observed atherosclerosis, see Di Bisceglie, etc., Hepatology (Hepatology 11: 764-772 (1990), Ameis, etc., J Lipid Res. (The study of lipids) 36: 241-250 (1995). Manifestation BNAH very changeable, and in some patients the disease is not diagnosed before the onset of complications in late adulthood, while in other patients there is a liver dysfunction in early childhood. BIH is associated with a reduced life expectancy and significant deterioration of health. Life expectancy of people with BIH depends on the severity of disease-related complications.

Currently, the methods of treatment of Wolman disease is very limited. Infants with hyperthermia and/or signs of infection with antibiotics. Can be assigned leroidesanimaux therapy for adrenal insufficiency and special Parente�real food, and while there is no evidence that these measures prevent the onset of death, it is unclear whether they have any impact on short term survival. In a series of four patients with a deficiency of CFLs, in the treatment using bone marrow all four died due to complications within a few months after the transplant. Although some success has been described in subsequent histories, the mortality rate remains high, and many patients transplantation is not performed because of their very poor condition and a low probability of survival in preparation for transplantation. A very small number of centenarians reported, indicates that the correction of the enzyme deficiency in hematopoietic cells is a sufficient condition for significant improvement of the clinical condition in this disease. Usually clinical support is provided through dietary restrictions when trying to limit the accumulation of non-transportable and non-degradable lipids associated with acute manifestations of the disease, leading to death.

Currently, the methods of treatment of phenotype BIH focused on symptomatic therapy through monitoring the accumulation of lipids by diet that excludes foods high in cholesterol and triglyceride�in, and suppress the synthesis of cholesterol and formation In apolipoprotein using prescription drugs that lower cholesterol levels (eg, statins and cholestyramine). Although it is possible to observe some clinical improvement, underlying the existence of the disease remains, and the disease continues.

It was suggested that enzyme replacement therapy using recombinant CFLs may be a viable method of treating deficiency of acid lysosomal lipase and related conditions (see Meyers and others (1985) Nutrition Res. (Study power) 5(4):423-442; WO 9811206; and Besley (1984) Clinical Genetics (Clinical genetics) 26:195-203). Some studies deficit CFLs using mice showed correction of some anomalies in mice with deficiency of CFLs (CFL-/-) at introduction of high doses (more than 1 milligram per 1 kilogram of body weight) of recombinant human CFLs once every 3 days (see, e.g., Grabowski US 2007/0264249). In the earlier studies for the correction of deviations in mice with deficiency of CFLs was assumed that relatively large quantities and frequent administration of recombinant protein CFLs are required for correction of the underlying phenotypes. It is also important to note that unlike models using rats with CFL-/-and originally described by Yoshida and Kuriyama (1990) Laboratory Animal Science (And�adherence to laboratory animals), volume 40, pp. 486-489, model using mice with CFL-/-used in the studies above, has close similarities with BV people in the sense that the deficit CFLs in mice leads to impaired growth observed in humans.

To date, exogenous CFLs were not prescribed to the people and effective therapy available for the treatment of deficiency of CFLs, including BV, BIH and other diseases. Therefore, there is an acute need for methods of treatment with reduced frequency of drug administration to improve the quality of life of patients. Additionally, preferably, the determination of therapeutically effective doses to restore growth, normalization of liver function, increase in the concentration of CFLs in tissues and increasing the activity of CFLs in sick people.

Summary of the INVENTION

The present invention based on the first clinical cases where patients were successfully administered dose of exogenous CFL. The baby is suffering from a deadly form of deficit CFL (Wolman disease or early stage of deficiency CFL), was effectively cured through the introduction of exogenous CFLs, and safety evaluation of substitute therapy using enzyme CFL was performed on a group of human patients suffering from a late form of scarcity CFL. The infant with early-onset deficiency CFLs were introduced weekly low-dose �without showing any adverse effects or reactions. Drastic improvement of the main indicators of the state of the organism and clinical/laboratory tests of the efficiency was observed after one to two weeks after the initial injection. After 4 months of weekly doses of was restoring the normal growth of the baby and significant improvement in all indicators related to the deficiency of CFLs, including malabsorbtsija, hepatomegaly and liver function. Adult patients with advanced form of the disease is also applied weekly dose with a small amount of exogenous CFLs with no signs of adverse effects. Thus, the clinical data collected to date show that enzyme replacement therapy with exogenous CFLs present invention provides a safe and effective treatment of deficiency of CFLs.

Accordingly, the present invention provides methods of treating diseases or conditions associated with a deficiency of CLL patients-people, through the introduction of an effective amount of exogenous acidic lysosomal lipase (CFL). Exogenous CFLs can be a recombinant CFLs person having the structure N-linear glycan containing at least one mannose and/or mannose-6-phosphate. Exogenous CFLs effectively internalized into the lysosome, e.g., lymphocytes, macrophages and/or fibroblasts.

In not�representing variants of an embodiment of a patient, suffering from shortage of CFLs, is diagnosed with Wolman disease (WD). In one embodiment, the introducing is sufficient to enhance the growth of a patient with BV. In one embodiment, the introducing is sufficient to restore normal growth of the patient with VWD. In other embodiments, the embodiment of a person suffering from deficiency of CFLs, the disease is diagnosed the accumulation of cholesterol esters (BNAH). Methods of treatment in accordance with the present invention can be applied to patients-for people of any age.

In this application also provides methods of treating a patient suffering from a deficit of CFLs, the introduction of he recombinant CFLs human in an effective amount to improve liver function. In some embodiments of the incarnation introduction is sufficient to normalize the test results of the liver. In one embodiment, the introducing is sufficient to reduce the levels of liver transaminases. For example, liver transaminases may contain whey aspartataminotransferaza (ACT) and/or serum of alanineaminotransferase (ALT). In one embodiment, the introduction is enough to minimize hepatomegaly. In one embodiment, the introducing is sufficient to reduce the size of the liver of the patient. In one embodiment, the introduction is sufficient� to reduce the levels of serum ferritin blood.

In one embodiment, the introducing is sufficient to reduce the levels of serum ferritin blood, including, for example, the levels of cholesterol ester (CE) and/or triglycerides (TG).

Is also provided a method of increasing the activity of CFLs in a patient with deficiency of CFLs. This method includes the introduction of recombinant CFLs human patient to enhance the activity of CFLs, which can be determined, for example, in lymphocytes and/or fibroblasts.

In one embodiment describes a method for the treatment of a condition associated with deficiency of CFLs, in a patient by administration of an effective amount of exogenous protein CFLs patient from once every 5 days to once every 30 days.

In some embodiments embodiment the patient suffering from deficiency of CFLs, is entered dose from about 0.1 mg to about 50 mg of exogenous CFLs per kilogram of body weight. In one embodiment, the patient is injected a dose from about 0.1 mg to about 10 mg of exogenous CFLs per kilogram of body weight. In one embodiment, the patient is injected a dose from about 0.1 mg to about 5 mg of exogenous CFLs per kilogram of body weight.

In one embodiment, the dose of administration is from about 0.1 mg/kg/HR to about 4 mg/kg/h.

In some embodiments embodiment, the patient is treated using the second drug environments�TBA. The second medicament may include, for example, an agent for lowering cholesterol (e.g., a statin or ezetimibe), an antihistamine (e.g., diphenhydramine) or immunosuppressant.

BRIEF description of the DRAWINGS

Figure 1A shows the serum levels of aspartate aminotransferase baby boy with Wolman disease (i.e. an early form of deficit CFL) who received a weekly dose of exogenous CFL (SBC-102) (dose: 0.2 mg/kg (initial introduction; week 0); 0.3 mg/kg (week 1); 0.5 mg/kg (week 2); and 1.0 mg/kg (weeks 3-8)). Figure 1B shows the levels of serum alanine aminotransferase (ALT) in the same patient. The age of the patient at initial introduction was 4 months and 1 week.

Figure 2 shows the levels of ferritin in the serum of a patient with Wolman disease who received a weekly dose of exogenous CFL (SBC-102) (dose: 0.2 mg/kg (initial introduction; week 0); 0.3 mg/kg (week 1); 0.5 mg/kg (week 2); and 1.0 mg/kg (weeks 3-8)). The age of the patient at initial introduction was 4 months and 1 week. The levels of serum ferritin blood is shown from week 1 after the initial dose in week 0.

Figure 3 shows the growth rate of a patient with Wolman disease who received a weekly dose of exogenous CFL (SBC-102) (dose: 0.2 mg/kg (initial introduction; week 0); 0.3 mg/kg (weeks�La 1); 0.5 mg/kg (week 2); and 1.0 mg/kg (weeks 3-8)).

Figure 4 shows a graph of the growth of the patient with Wolman disease (kg, % weight at age for boys).

Figure 5 shows the serum levels of aspartate aminotransferase 41-year-old white male with BIH who received weekly doses of 0.35 mg/kg of exogenous CFL.

Figure 6 shows serum levels (ALT) in 41-year-old white male with BIH who received weekly doses of 0.35 mg/kg of exogenous CFL.

Figure 7 shows the serum albumin levels in 41-year-old white male with BIH who received weekly doses of 0.35 mg/kg of exogenous CFL.

Figure 8 shows the levels of serum ferritin blood 41-year-old white male with BIH who received weekly doses of 0.35 mg/kg of exogenous CFL.

Figure 9 shows the rate of increase in weight in four male rats of the same age, each of which was administered exogenous CFLs in accordance with one of the following modes of administration: 1 mg per kilogram once a week, 5 mg / kg once a week, 5 mg / kg once every 2 weeks or placebo. The numbers in the columns show the age in days.

Figure 10 shows the results of pathological and histopathological studies of rats with deficiency of CLL after treatment with exogenous CFLs and rats with deficiency of CLL after treatment with placebo. Makr�pathological study showed a normalization of the color and size of the liver in rats after treatment with exogenous CFL. Histopathological examination of the liver tissue of rats after treatment with exogenous CFLs showed essentially normal histology of the liver differs sharply from significant accumulation of foamy macrophages in animals injected with placebo.

Figure 11 shows colocalization recombinant CFL (SBC-102) and the lysosomal marker in the lysosomes of cells examined by fluorescence microscopy method using sequential scan mode

Figure 12 shows the specificity of the Association of recombinant CFL (SBC-102) with the receptor N-acetylglucosamine/mannose measured by competitive binding analysis using cell lines of macrophage, NR8383.

Figure 13 shows the activity of recombinant CFLs person in normal cells and cells with deficiency of CFLs, in vitro.

Figure 14 shows the effect of treatment with the introduction of recombinant CFLs person on the weight of the internal organs of rats with deficiency of CFLs. The size of the bodies presented as a percentage of body weight, determined at the age of 8 weeks, rats CFL-/-and CFL+/+after weekly administration environment or SBC-102 in an amount of 5 mg/kg for 4 weeks.

Figure 15 shows the body weight in wild type and rats with a deficit after the introduction of CFLs environment or SBC-102 in an amount of 5 mg/kg-1within 4 weeks. The dose �provided on the X-axis diamonds, since 4 weeks.

Figure 16 shows the levels of cholesterol, cholesterol ester and triglyceride in the liver defined at the age of 8 weeks in rats with BV and deficit CFL weekly after the introduction of the environment or SBC-102 in an amount of 5 mg/kg-1within 4 weeks.

Figure 17 shows the increase in body weight in percentage of rats with deficiency of CFLs.

Figure 18 shows liver weight as percentage of body weight in rats with deficiency of CFLs after the administration of SBC-102 for 4 weeks.

Figure 19 shows the levels of cholesterol esters in tissues of rats with deficiency of CFLs after the administration of SBC-102 for 4 weeks.

DETAILED description of the INVENTION

The present invention provides methods of treating a person suffering from a disease or condition that is sensitive to the introduction of exogenous acidic lysosomal lipase.

Definition

For convenience, certain terms used in the description, examples and appended claims are formulated in this application to illustrate and define the meaning and scope of various terms used to describe the present invention.

"CFL" in accordance with the usage in this application refers to "acid lysosomal lipase", and the two terms are used interchangeably in the description. CFLs can be a human protein, i.e. an acidic lizaso�ing the human lipase. The term "SBC-102" in accordance with the usage in this application relates to recombinant acidic lysosomal lipase of human rights. The term of CFLs also apply to such substances, presented in the literature, as the acid hydrolase cholesterolemia ether, granulocyte esterases cholesterol, Lipase A, LINDEN A, and granulocyte esterases of Sterol.

CFL catalyzes the hydrolysis of cholesterol esters and triglycerides to free cholesterol, glycerol and free fatty acids. Thus, "activity CFLs" can be measured, for example, like splitting fluorogenic substrate, 4-methylumbelliferyl oleate (MWA). Splitting MWA can be detected, for example, by excitation at a wavelength of 360 nm and emission at 460 nm of the free fluorophore, 4-methylumbelliferyl (MU). The results can be presented in relative fluorescence units (OEF). For example, the amount of substrate cleaved 30-minute study on the endpoint, can be quantified relative to a reference curve MW, and one unit (E) activity can be defined as the amount of enzyme required for the breakdown of 1 micromole MWA per minute at 37°C. Accordingly, the functional fragments of the variants of CFLs include fragments containing the activity of CFLs, for example, the ability to hydrolyze cholesterol esters and/or triglycer�s.

In accordance with the usage in this application "exogenous CFL" refers to CFLs, which is not produced to patients in a natural way. For example, exogenous CFL includes a recombinant protein of CFLs, which is administered to the patient, the protein CFL, which is excreted in human or animal and injected to the patient, and protein CFL secreted (i.e., expressed) in a patient resulting from the introduction of CFLs-coding RNA and/or DNA or other treatment that increases the expression of exogenous protein CFL.

"Intravenous injection" is often regarded as an intravenous bolus or rapid injection of doses of a substance and refers to the method of administration, when the syringe is connected to the device access to the vein and the drug is injected directly, usually fast, but sometimes up to 15 minutes if the introduction can lead to irritation of the vein or the effect of too rapid administration. After the introduction of the drug into the device needed some means to ensure its introduction into the patient. Usually this is achieved by ensuring the free flow of the liquid medium carrying the drug into the blood stream. However, in some cases after the first injection the second injection is used a liquid medium, often called "brainwashing", to facilitate the entry of drugs into the blood stream.

"Votive�Noah infusion" refers to the method of administration, in which the drug is administered over an extended period of time. For example, the drug may be administered to the patient within 1 to 8 hours. The drug also may be administered to the patient within about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 hours. For the implementation of the intravenous infusion may be used IV fluids for injection by gravity or pump for intravenous infusion. Intravenous infusion is usually used in those cases when a patient requires administration of the drug only during certain periods of time and does not require the introduction of additional substances (for example, aqueous solutions which may contain sodium chloride, glucose or any combination), which restore the level of electrolytes, blood sugar, to compensate for water losses.

The term "bird" in accordance with the usage in this application refers to any species, subspecies or race of organism of the birds of the taxonomic class, such as, but limited to, chickens, turkeys, ducks, geese, quail, pheasants, parrots, finches, hawks, crows and beskrive birds, including ostriches, EMUs and cassowaries. The term includes the various known race Gallus gallus, or chickens, (for example, white Leghorn, brown Leghorn, striped rock, Sussex, new Hampshire, Rhode island, the a $ �RP, Minorca, amrox, California gray), as well as race, turkeys, pheasants, quails, duck, ostriches and other poultry, commonly bred in commercial quantities. The term also includes an individual organism birds at all stages of development, including embryonic and fetal condition.

The term derived from poultry or derived from birds" refers to a composition or substance produced by or derived from poultry. The term "poultry" refers to the birds that can be kept as Pets, including but not limited to, chickens, ducks, turkeys, quails and basilevych birds. For example, the term "obtained from poultry" can refer to a substance obtained from chickens obtained from Turkey and/or derived from quail.

The term "patient" in accordance with the usage in this application refers to any person who receives, or has received, or will receive medical care or treatment, for example, in accordance with the instructions of the health care provider.

The term "therapeutically effective dose" in accordance with the usage in this application refers to the dose (e.g., number and/or interval) of the drug, which is supposed to provide the intended therapeutic response. A therapeutically effective dose Rel�the necessity of the dose, the result of the introduction of which when compared with a corresponding subject who has not received such a dose is the improvement of the process of treatment, cure, prevention or reduction of intensity of the disease, disorder, or side effect, or a reduction in the frequency of symptoms, or prevention of disease or disorder. The term also includes within its borders the use of dose that is effective for improving physiological functions.

The terms "treat", "treatment" and "treatment" refer to methods of alleviating, mitigating or improvement of the disease or its symptoms, preventing additional symptoms, mitigate, or prevent the underlying causes of symptoms, suppression of the disease or condition, relieving the disease or condition, relieving the disease or condition, encouraging a return of the disease or condition, relieving a condition caused by a disease or condition or preventing disease symptom or condition prophylactically and/or after onset of symptom.

In accordance with the usage in this application, with a specific dose "kg-1", "kg", "/kg" and "kilo" means "per kilogram of body weight of a mammal, and these terms can be used interchangeably.

In accordance with the usage in this application the termi� "polypeptide" is intended to cover "polypeptide" in the singular, and "polypeptides" is plural and refers to a molecule composed of monomers (amino acids) linearly linked amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "chains of amino acids" or any other terms used for the chain or chains of two or more amino acids included within the term "polypeptide" and the term "polypeptide" may be used instead of any of these terms are interchangeable. The term "polypeptide" is also intended for product modification polypeptide after expression, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization known protecting/blocking groups, proteolytic cleavage, or modification of the unnatural amino acid of origin. The polypeptide may be derived from a natural biological source or produced using recombinant technology, but not necessarily by translation from the designated nucleic acid sequence. It can be generated by any method, including chemical synthesis.

In accordance with the use in the d�the auditors request the relative homology between two amino acid sequences or two nucleotide sequences is equivalent to the relative identity of the two sequences. The relative identity of two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = numbers of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of two sequences. Comparison of sequences and determination of the relative identity of two sequences can be accomplished using a mathematical algorithm, as described in the examples below, non-exhaustive example.

The relative identity of two amino acid sequences can be determined using the algorithm of Meyer and Miller (E. Meyers and W. Miller (Comput. Appl. Biosci, 4:11-17 (1988)), which was introduced into the ALIGN program (version 2.0), using the weight table for the remainder of the RAM 120, the ratio of the length of the gap 12 and the ratio of the gap 4. In addition, the relative identity of two amino acid sequences can be determined using the algorithm of Needleman and Vanšu (Needleman and Wunsch (J. Mol, Biol. 48:444-453 (1970)), which was introduced into the GAP program of the software package GCG (http://www.gcg.com), or using a matrix of Blossom 62 or matrix RAM and the weight ratio of the gap 16, 14, 12, 10, 8, 6, or 4 weighting length 1, 2, 3, 4, 5 or 6.

"From�graded" polypeptide or fragment, variant or derivative product is assigned to a polypeptide that is not found in the natural environment. Specific level of treatment is required. For example, an isolated polypeptide can be extracted from native or natural environment. Produced recombinante polypeptides and proteins expressed in the cells of the host are considered as isolated, as disclosed in this application, as either native or recombinant polypeptides which have been separated, fractionated, or partially or mostly cleaned by any suitable method.

Other polypeptides disclosed in this application, represent fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination of them. The terms "fragment", "variant", "derivative" and "analog" when referring to any of the polypeptides disclosed in this application include any polypeptides which retain at least some activity of the corresponding native polypeptide (e.g., fragments, variants, derivatives and analogs of polypeptides of CFLs that retain the ability to hydrolyze cholesterol esters and/or triglycerides). Fragments of the polypeptides include, for example, proteolytic fragments, and fragments of the deletions. Variants of polypeptide include fragments as described above, as well as �polypeptide with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Options may have a natural or unnatural origin. Options unnatural origin can be produced using known methods of mutagenesis. A variant polypeptide may include conservative or non-conservative amino acid substitution, deletion or insertion. Derivatives are polypeptides which have been modified to the appearance of additional properties that are absent and native polypeptides. Examples include hybrid proteins. Variant polypeptides can also be referred to as "polypeptide analogs". In accordance with the usage in this application, the "derivative" of the original polypeptide can contain one or more residues obtained chemically by reaction of a functional side group. To "derivatives" include peptides containing one or more amino acid derivative naturally occurring twenty standard amino acids. For example, 4-hydroxyproline may be substituted for Proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine can be replaced by histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

The term "polynucleotide" is intended to encompass nucleic acids in the singular, as well as nucleic frequencies in m�austinom and refers to an isolated molecule or construct, nucleic acid, for example, the messenger RNA (mRNA) or plasmid DNA (pdnk). The polynucleotide can include traditional fosfodiesterazu communication or alternative communication (e.g., an amide bond, such as found in peptide nucleic acid (NCP). The term "nucleic acid" refers to any one or more segments of nucleic acid, such as DNA fragments or RNA present in the polynucleotide. "Isolated" nucleic acid or "isolated" nucleotide is a molecule of nucleic acid, DNA or RNA, which was extracted from their own environment. For example, a recombinant polynucleotide encoding CFLs contained in the vector is considered as isolated for purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides contained in heterologous cells-owners, or purified (partially or mostly) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides according to the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules, obtained artificially. In addition, the polynucleotide or nucleic acid may be a regulatory� element or to include such an element, for example, the promoter, the binding site of ribosomes or transcription terminator.

In accordance with the usage in this application "coding region" is a portion of nucleic acid comprising codons that are translated to amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated to amino acids, it may be regarded as part of the coding region, but any flanking sequences, for example promoters, the sites of binding of ribosomes, transcription terminators, introns and the like, are not part of the coding region. Two or more coding region in accordance with the present invention can be presented in one polynucleotide constructs, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on a separate (different) vectors. In addition, any vector may contain one encoding region or may include two or more coding region. In addition, a vector, polynucleotide, or nucleic acid in accordance with the present invention may encode heterologous coding region, fused, or nalivshiesya with nucleic acid that encodes a polypeptide of CFLs or a fragment, variant or derivative from them. Heterologous coding region include without limitation special elements or motifs, that�s as a secretory signal peptide or a heterologous functional domain.

Various management transcription known to specialists in this field. These include, without limitation, the management of transcription, functioning in vertebrate cells, such as, but not limited to, segments of the promoter and enhancer with cytomegalovirus (immediate early promoter, in conjunction with itroom-A), aqualicious simian virus 40 (the early promoter), and retroviruses (such as the rous sarcoma virus). Other areas of management include transcription region derived from vertebrate genes such as actin, heat shock protein, growth hormone, bovine, B-globin rabbit, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional appropriate management transcription include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

Similarly, the various controls transcription known to specialists in this field. These items include, but are not limited to, the sites of binding of ribosomes, codons for the initiation and termination of translation, and elements derived from picornaviruses (particularly an internal site of binding of ribosomes (WOSR), also called the pic�egovernance CITE).

In other embodiments, embodiments of the invention the polynucleotide in accordance with the present invention is an RNA, for example, in the form of the messenger RNA (mRNA).

The encoding region of the polynucleotide and the nucleic acid in accordance with the present invention may be associated with additional coding regions which encode secretory or signal peptides which direct the secretion of the polypeptide encoded by the polynucleotide in accordance with the present invention. In accordance with the hypothesis of a signal sequence, proteins that are excreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the Mature protein after initiation of the export of the growing protein chain across the granular endoplasmic reticulum. Specialists in this field know that the polypeptides synthesized by vertebrate cells generally have a signal peptide fused with the N-end of the polypeptide, which is cleaved from the complete or primary polypeptide for the production of secretory or Mature form of the polypeptide. In certain embodiments, the realization of his own signal peptide, for example, uses a signal peptide MKMRFLGLVVCLVLWTLHSEG (SEQ ID NO:2) CFL man or a functional derivative of that sequence that retains �the ability to direct the secretion operably associated polypeptide. Alternative, can be used heterologous signal peptide (e.g., a heterologous signal peptide mammals or birds) or its functional derivative. For example, leading the wild-type sequence may be replaced by plasminogen activator tissue (PTC) or β-glucuronidase mouse.

The term "vector" means a polynucleotide consisting of single-stranded, double-stranded, circular or overspilling DNA or RNA. A typical vector may consist of the following elements that are functionally associated and located at certain distances to allow for functional gene expression: the starting point of replication, a promoter, an amplifier, a leader sequence 5' mRNA, the binding site of ribosomes, nucleic acid cassette, sites, termination and polyadenylation and a selectable marker sequence. One or more of these elements may be omitted in specific applications. The nucleic acid cassette may include a restriction site for inserting a nucleic acid sequence that needs to be expressed. Functional vector the nucleic acid cassette contains a nucleic acid sequence that needs to be expressed, including sites of translation initiation and termination. The design can be further on�Chen intron, for example, 5 in the encoding sequence. The vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence relative to the control sequence, which coding sequence is transcribed under the "control" of the management or regulatory sequence. Preferably, the modification of the sequences encoding a particular protein, achieved this end. For example, in some cases, you may need to modify the sequence so that it could be attached to the control sequences with the appropriate orientation, or to preserve the reading frame. Control sequences and other regulatory sequences can be joined to the coding sequence prior to insertion into the vector. Alternative encoding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site in reading frame and under the regulatory control sequences.

The term "expression" in accordance with the usage in this application refers to PR�the process, by which a gene produces a biochemical product, e.g. the polypeptide. The process includes all of the functional manifestations of the presence of a gene in a cell, including, without limitation, gene knockdown, as well as transient and stable expression. The process includes without limitation transcription of the gene in the messenger RNA (mRNA) and translation of such mRNA into polypeptide (s). Gene expression produces a "gene product". In accordance with the usage in this application gene product may be a nucleic acid, for example, the messenger RNA produced by transcription of a gene or polypeptide that is transmitted from the transcript. Gene product described in this application additionally includes nucleic acids with post-transcriptional modifications, such as polyadenylation or polypeptides with post-translational modifications, such as methylation, glycosylation, addition of lipids, Association with other protein subunits, proteolytic cleavage, etc.

In accordance with the usage in this application, the term "the host cell" refers to cells that constitute the main vector using recombinant DNA encoding at least one heterologous gene.

In accordance with the usage in this application, the terms "N-Gly�EN", "oligosaccharide", "the structure of the oligosaccharide chains", "glycosylation pattern", "glycosylation profile" and "structure of glycosylation" is largely a single value, and each of them refers to one or more structures formed from sugar residues and attached to glycated proteins.

In accordance with the usage in this application, the term "pharmaceutical composition" refers to a mixture of the composition described in this application, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspendida agents, thickeners and/or fillers.

Patients with insufficient activity of CFLs

Without the desire to limit the invention to the treatment of any state or group of States of the invention includes treatment of deficiency of acid lysosomal lipase (CLL) patients. In accordance with the usage in this application the patient with deficiency of CFLs is any patient with insufficient activity of CFLs. Insufficient activity of CLL patients may be the cause of low RNA levels, low protein or low activity of proteins. Insufficient activity of the CFL may be the result of mutations in the sequence encoding the CFL, regulatory sequences CFLs or another gene (e.g., gene regulatory CFL). Insufficient actively�th CFLs can also be the result of environmental factors.

The present invention can be used to treat a wide range of conditions in a subject or patient. Therefore, any condition that can be successfully treated by application of exogenous CFL in accordance with the invention is included in the scope of the invention.

One variant of an embodiment of the invention relates to the treatment of lysosomal storage diseases (LSD) resulting from deficiency of acid lysosomal lipase, particularly that of Wolman disease (WD) and diseases of the accumulation of cholesterol esters (BNAH). Without the desire to limit the invention to any particular theory or by a private mechanism of action, and BV and BIH can be the result of mutations in the locus of CFLs and lead to massive accumulation of lipid material in the lysosomes in a number of tissues, and significant impairment in cholesterol and lipid homeostatic mechanisms that can be cured by the introduction of exogenous CFL in accordance with the methods of the present invention. Thus, in one embodiment, the deficit CFLs, which are treated in accordance with the invention, is a BV. In another embodiment, the deficit CFLs, which are treated in accordance with the invention, is BIH. In some embodiments embodiment, the diagnosis of BV and BIH based on genetic analysis (e.g., identification�Oia functional mutations in the sequence coding CFL). In other embodiments embodiment, the diagnosis of BV and BIH is based on clinical data (e.g., physical examination and/or laboratory tests).

In some embodiments, embodiments of exogenous CFLs can be used to treat complications associated with various conditions, such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (Nash). NAFLD refers to a liver disease with histopathology similar histopathology liver disease arising from excessive alcohol consumption. It is characterized macrovascular obesity, leading to an increase in the liver. NAFLD can progress to Nash, which refers to liver disease, such NAFLD with the addition of inflammation and liver damage that can lead to fibrosis and cirrhosis.

In some embodiments, embodiments of exogenous CFLs can be used to treat conditions including pancreatitis, such as chronic pancreatitis and/or acute pancreatitis, damage to the pancreas caused by alcohol, such as alcohol induced pancreatitis.

Exogenous CFLs produced by any suitable method, may be used to treat diseases caused by damage to cells caused by alcohol, including, but not limited to, such induced alcoholimpaired cells which lead to accumulation of lipid esters in the tissues of the body, such as, but not limited to, liver, spleen, digestive tract and cardiovascular tissue. In accordance with the invention malabsorption can also be treated by introducing exogenous CFL. Exogenous CFLs is also useful in the treatment of patients with Tangier disease and hereditary hypoalphalipoproteinemia. Tangier disease/hereditary hypoalphalipoproteinemia is associated with accumulation of cholesterol esters in macrophages and is accompanied by hepatosplenomegaly and/or lymphadenopathy together with low levels of high density lipoprotein (HDL), which can be treated by introducing exogenous CFL. For example, without the desire to limit the invention to any particular theory or mechanism of action, decreased activity of CFLs may lead to reduced expression AVSA, and, conversely, increased activity of CFLs obtained by introduction of exogenous CFLs patient with Tangier disease hereditary hypoalphalipoproteinemia will increase expression of AWSA to overcome the effects of the gene AWSA with reduced functional activity as a result of polymorphism.

In some embodiments embodiment the level of activity of CFLs in the patient before treatment is about 1%, about 2%, about 3%, about 5%, PR�approximately 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80% of the normal level of activity CFLs. In one embodiment, the activity level of CFLs in the patient before treatment is about 50% or less of the normal level of activity CFLs. In one embodiment, the activity level of CFLs in the patient before treatment is approximately 40% or less of the normal level of activity CFLs. In some embodiments embodiment the level of activity of CFLs in the patient before treatment is approximately 30% or less of the normal level of activity CFLs. In some embodiments embodiment the level of activity of CFLs in the patient before treatment is approximately 20% or less of the normal level of activity CFLs. In some embodiments embodiment the level of activity of CFLs in the patient before treatment is approximately 10% or less of the normal level of activity CFLs. In some embodiments embodiment the level of activity of CFLs in the patient before treatment is approximately 5% or less of the normal level of activity CFLs. In some embodiments embodiment the patient before treatment is not detectable measured activity level CFL.

In some embodiments embodiment, the activity level CFL is measured in cultured fibroblast obtained from the patient-the person �tradego deficit CFL. In some embodiments embodiment, the activity level CFL is measured in lymphocytes (e.g., leukocytes) patient-a person suffering from a deficit of CFLs. Lymphocytes include, but are not limited to, peripheral blood mononuclear cells (PBMCs). Methods of measurement are described, for example, Burton et al., (1980) Clinica Chemie Acta 101: 25-32, and in Anderson et al., (1999) Mol. Genet. & Metab., 66: 333 - 345; both sources included in this application in its entirety. Patients with deficiency CFL receiving treatment through the introduction of exogenous CFLs, can demonstrate the enzyme activity fibroblast CFL, which is below about 30, about 20, about 10, about 5, about 4, about 3, about 2, or about 1 pmol/mg/min when measured with the use of triolein the substrate. Patients with deficiency CFL receiving treatment through the introduction of exogenous CFLs, can demonstrate the enzyme activity of leukocyte CFL, which is below about 30, about 20, about 10, about 5, about 4, about 3, about 2, or about 1 pmol/mg/min when measured with the use of triolein the substrate. Patients with deficiency CFL receiving treatment through the introduction of exogenous CFLs, can demonstrate the enzyme activity fibroblast CFL, which is below about 30, about 20, about 10, about 5, about 4, about 3, about 2 or prima�but 1 pmol/mg/min when measured with the use of cholesterol oleate as substrate. Patients with deficiency CFL receiving treatment through the introduction of exogenous CFLs, can demonstrate the enzyme activity of leukocyte CFL, which is below about 30, about 20, about 10, about 5, about 4, about 3, about 2, or about 1 pmol/mg/min when measured with the use of cholesterol oleate as substrate.

The introduction of exogenous CFL

The invention provides methods for the treatment of human patients by exogenous CFLs, including the introduction of exogenous CFLs patient, where the introduction is sufficient to restore growth, improve liver function, reduce liver damage, increase the level of CFLs in the tissues and/or increase the activity of CFLs in the patient. The invention provides an effective and previously characterized the frequency of administration (i.e., graphs of doses) exogenous CFLs for treating conditions resulting from deficiency of CFLs, including BV and BIH, and previously characterized the number of dosing for treatment of these conditions.

The invention provides a therapeutically effective dose of exogenous CFLs, which must be administered to the patient once every 5 days to once every 30 days during the period of time determined by a specialist practitioner in the field of medical science. In one embodiment, the time period will be the remaining PE�iodine the patient's life. In one embodiment, the frequency of the dose is in the range from once every 5 days to once every 25 days. In one embodiment, the frequency of the dose is in the range from once every 5 days to once every 21 days. In another embodiment, the frequency of the dose is in the range from once every 7 days to once every 14 days. Exogenous CFLs may be administered 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, embodiments of exogenous CFL is injected about once a week. In other embodiments, the embodiment of exogenous CFL is entered once in about two weeks. In one embodiment, the exogenous CFL is entered once approximately every 30 days.

For treatment of the condition, as a rule, the amount of exogenous CFLs may vary depending on known factors such as age, health and weight of the recipient, kind of concurrent treatment, frequency of treatment, etc. the Usual dose of active ingredient may be equal to from about 0.01 to about 50 mg per kilogram of body weight. In one embodiment, the dose of exogenous CFL in accordance with the Fig�the group is from about 0.1 mg to about 0.5 mg per kilogram of body weight. In one embodiment, the dose is from about 0.1 mg to about 5.0 mg per kilogram. In one embodiment, the dose is from 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 to about 0.45, and approximately 0,50 mg / kg. In one embodiment, the dose is from 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 / kg. For example, can be injected 0.1 mg per kilogram of body weight, 0.2 mg / kg 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, 1 mg per kilogram of body weight, 2 mg per kilogram of body weight, 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 from about 1 mg to about 20 mg per kilogram of body weight.

The invention also includes other dose when using graphics a dose in accordance with the invention. For example, in accordance with the schedule of doses in accordance with the invention, the patient is from about 0.1 mg to about 50 mg per kilogram of body weight.

In some embodiments of the incarnation �usual from about 0.5 to about 50 mg of exogenous CFL, for example, the patient with Wolman disease at the age from 1 month to 24 months. In one embodiment, the patient's age is less than 1 year. In another embodiment, the patient's age is less than 2 years. In some embodiments embodiment 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 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg or about 45 mg of exogenous CFL is administered to a patient with Wolman disease. In some embodiments, embodiments from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, or from about 0.5 mg to about 5 mg inserted into a patient with Wolman disease. In some embodiments of the incarnation is introduced from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, or from about 1 mg to about 5 mg.

In some embodiments, embodiments from about 1 mg to about 350 mg of exogenous CFL is administered to a patient with BENCH. Thus, in some embodiments embodiment about 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 mg of exogenous CFL is administered to a patient with BENCH. In some embodiments, embodiments from about 5 mg to about 350 mg, from about 5 mg to about 300 mg, from about 5 mg �about 250 mg, from about 5 mg to about 200 mg is administered to a patient with BENCH. In some embodiments, embodiments from about 10 mg to about 350 mg, from about 10 mg to about 300 mg, from about 10 mg to about 250 mg, from about 10 mg to about 200 mg is administered to a patient with BENCH.

Comprehensive treatment

Therapeutic proteins disclosed in this application can be used in combination with other therapeutic agents. The invention provides a procedure performed before treatment to minimize or prevent any potential anaphylactic reactions that can occur when exogenous CFL in accordance with the invention. In one embodiment, to prevent possible anaphylactic reactions receptor antagonist N-1, also known as an antihistamine (e.g., diphenhydramine), is administered to a patient. In one embodiment, the receptor antagonist N-1 is introduced in the form of a dose from about 1 mg to about 10 mg per kilogram of body weight. For example, an antihistamine may be administered in an amount of about 5 mg per kilogram. The introduction of antihistamine can be performed before the introduction of exogenous CFL in accordance with the invention. In one embodiment, the receptor antagonist N-1 is introduced from about 10 to about 90 minutes, for example from about 30 to about 60 minutes prior to administration aksogan�th CFL. Antagonist of receptor N-1 can be administered using an ambulatory system connected to the vascular access port. In one embodiment, the antihistamine is administered in about 90 minutes prior to the introduction of exogenous CFL. In one embodiment, the antihistamine is introduced from about 10 to about 60 minutes prior to administration of exogenous CFL. In another embodiment, the antihistamine is introduced from about 20 to about 40 minutes before the introduction of exogenous CFL. For example, an antihistamine may be administered at 20, 25, 30, 35, or 40 minutes prior to the introduction of exogenous CFL. In one embodiment, the input is an antihistamine diphenhydramine. You can use any suitable antihistamine. Such antihistamines include, without limitation, clemastine, doxylamine, loratidine, desloratidine, Fexofenadine, Pheniramine, cetirizine, ebastine, promethazine, chlorphenamine, levocetirizine, olopatadine, quetiapine, meclizine, dimenhydrinate, ambreen, cimetidin and dexchlorpheniramine.

In one embodiment, the antihistamine is entered as a dose from about 0.1 mg to about 10 mg per kilogram of body weight. In one embodiment, the antihistamine is entered as a dose from 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. An antihistamine may introduces�Xia by any suitable method. In one embodiment, the antihistamine is administered intravenously. In another embodiment, the antihistamine is introduced into pharmaceutically acceptable capsules.

In another embodiment, the intravenous infusion of the likelihood of an anaphylactic reaction can be reduced by the introduction of drugs using a log linear slew rate. In this context, the ramp-up Protocol refers to a slow increase in the rate of infusion of the drug during the infusion to reduce the sensitivity of the patient for infusion of the drug.

Immunosuppressants, such as, but not limited to, antihistamines, corticosteroids, sirolimus, voclosporin, cyclosporine, methotrexate, antibodies directed to the receptor of IL-2, antibodies directed to the receptor of T-cell directed antibodies TNF-alpha proteins or compound (e.g., infliximab, etanercept or adalimumab), antigen 4 cytotoxic T lymphocyte (CTLA-4-Ig (e.g., abatacept), antibodies anti-OX-40 may also be entered before, during or after administration of exogenous CFLs, for example, if the patient is expected or had an anaphylactic reaction or an adverse immune response.

The invention also encompasses the treatment, comprising administering compositions containing an exogenous CFLs in combination with one or more substances, which lower the level hole�prunes (for example, inhibitors reductase 3-hydroxy-3-methylglutaryl-coenzyme A). Examples of such substances are non-restrictive, include: atorvastatin (Lipitor® and Torvast®), fluvastatin (Lescol®), lovastatin (Mevacor®, Altocor®, Altoprev®), pravastatin (Livalo®, Pitava®), pravastatin (Pravachol®, Selektine®, Lipostat®), rosuvastatin (Crestor®) and simvastatin (Zocor®, Lipex®).

The effects of the introduction of exogenous CFL

The present invention provides for the correction or normalization-related disease manifestations after treatment through the introduction of exogenous CFL. Clinical course (i.e., improvement) after the introduction of CFLs can be monitored using the appropriate method or the suitable procedure.

In some embodiments embodiment the exogenous CFL is sufficient to achieve the values of Cmax from about 200 ng/ml to about 1500 ng/ml In some embodiments of the incarnation, the introduction of exogenous CFL is sufficient to achieve the values of Cmax from about 200 ng/ml to about 1000 ng/ml In some embodiments of the incarnation, the introduction of exogenous CFL is sufficient to achieve the values of Cmax from about 200 ng/ml to about 800 ng/ml In some embodiments of the incarnation, the introduction of exogenous CFL is sufficient to achieve the Cmax values of approximately 200 ng/ml, about 300 ng/ml, approximately 400 ng/ml, about 500 ng/ml, about 600 ng/ml, about 700 n�/ml, approximately 800 ng/ml, about 900 ng/ml, about 1000 ng/ml to about 1250 ng/ml, or about 1500 ng/ml In some embodiments of embodiment (C) max achieved during the infusion.

In some embodiments embodiment the exogenous CFL is sufficient to achieve the half-life (t1/2) CFLs less than 40 minutes. In some embodiments embodiment the exogenous CFL is sufficient to achieve the half-life (t1/2) CFLs less than 30 minutes. In some embodiments embodiment the exogenous CFL is sufficient to achieve the half-life (t1/2) CFLs less than 20 minutes. In some embodiments embodiment the exogenous CFL is sufficient to achieve the half-life (t1/2) CFLs less than 15 minutes. In some embodiments embodiment the exogenous CFL is sufficient to achieve the half-life (t1/2) CFLs less than 10 minutes. In some embodiments embodiment the exogenous CFL is sufficient to achieve the half-life (t1/2) About CFLs 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 minutes.

In some embodiments, embodiments of exogenous CFLs increases the activity of CFLs in the patient. Activity CFLs can be enhanced, for example, in the liver, spleen, lymph nodes, aorta, peripheral blood leukocytes, and/or Phi�the fibroblasts of the skin. In some embodiments embodiment, the activity of CFLs measured in extracts of lymphocytes isolated from blood samples.

Exogenous CFLs can enhance the activity of CFL, 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-fold compared with the activity before the introduction of CFLs. Exogenous CFLs can enhance the activity of CFLs in at least about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20-fold compared with the activity before the introduction of CFLs. Activity CFLs can be assessed using methods known in this field, including, for example, tests using cholesterol [l-14C]oleate, triolein (glycerine) three [l-14C]oleate), p-nitrophenyl myristate or substrates 4-Muo (4 methylumbelliferyl oleate).

In one embodiment, the volume of the body and tissues and categorization are used to determine the improvement after the introduction of exogenous CFL in accordance with the invention.

In one embodiment, the clinical course for a function / liver lesions after the administration of exogenous CFLs controlled in time by the quantitative determination of blood transaminases such as aspartate aminotransferase (AC), and/or alanineaminotransferase (ALT), and/or other biomarkers, such as albumin, alkaline phosphatase and bilirubin (direct and total).

In one embodiment, the clinical course is monitored using the technology of image acquisition. For example, and without limitation, the technology of image acquisition may include ultrasound, CT scanning, magnetic resonance image and nuclear magnetic resonance spectroscopy.

In some embodiments embodiment the exogenous CFLs in the form of doses described in this application, is sufficient to restore growth and/or increasing the mass of the body of human patients. The introduction of exogenous CFLs can also increase the growth rate (i.e., increasing of body mass) of infants or children suffering from deficiency of CFLs with an early start. For example, the introduction of exogenous CFLs could increase the rate of increase of body weight, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400% or about 500% of the growth rate observed before the introduction. In some embodiments embodiment the exogenous CFL restores the normal rate of growth in children suffering from deficiency of CFLs with early onset (e.g., Wolman disease), aged prima�but 1 month to about 24 months. The term "normal" in this context refers to the normal growth rate for patients who receive treatment determined by a specialist practitioner in the field of medical science.

In one embodiment, for example, in the treatment of BV and BNAH or other manifestations of deficiency of CFLs, hepatomegaly weakens considerably when returning the size of the liver to the larger than normal size by an amount of from about 1% to about 60%. The term "normal" in this context refers to the liver of normal size patient received treatment, as determined by a qualified practitioner in the field of medical science. In one embodiment, the size of the liver is reduced to a value exceeding the normal size by an amount of from about 1% to about 50%. In another embodiment, the size of the liver is reduced to a value exceeding the normal size by an amount of from about 1% to about 40%. In one embodiment, the size of the liver is reduced to a value exceeding the normal size by an amount of from about 1% to about 30%. In another embodiment, the size of the liver is reduced to a value exceeding the normal size by an amount of from about 1% to about 20%. In another embodiment, the size of the liver is reduced to a value exceeding the normal size by an amount of from about 10% to about 20%. For example�EP, the size of the liver may be at 10%, 11%, 12% 13% 14%, 15%, 16%, 17%, 18%, 19%, or 20% more than the normal size. In yet another embodiment, the size of the liver is reduced to a value exceeding the normal size by an amount of from about 0% to about 10%. For example, the size of the liver may be at 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% more than the normal size of the liver.

Treatment by exogenous CFLs can also improve liver function. Thus, in some embodiments embodiment, the treatment by exogenous CFL is sufficient to restore normal liver function and/or normalization of liver tests. In some embodiments embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum liver transaminases, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, and/or at least about 90%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum liver transaminases at least about 40%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum liver transaminases at least about 50%. In one embodiment, the treatment by exogenous CFL is sufficient to decrease�Oia level of serum liver transaminases, at least about 60%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum liver transaminases at least about 70%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum liver transaminases, at least about 80%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum liver transaminases, at least about 90%.

In some embodiments, embodiments of liver transaminase levels is alanineaminotransferase (ALT). In one embodiment, the introduction of exogenous CFL is sufficient to reduce the level of serum ALT. For example, the introduction of exogenous CFLs can reduce the level of serum ALT, for example, at least about 50%, 60%, 70%, 80% or 90%. The level of serum ALT may serve to indicate the degree of liver damage. Thus, the present invention is also considering ways to reduce the degree of liver damage in a patient-a person suffering from a deficit of CFLs, through the introduction of an effective amount of exogenous CFLs to reduce the level of serum ALT.

In some embodiments, embodiments of liver transaminase levels is an aspartate�aminotransferase (ACT). In one embodiment, the introduction of exogenous CFL is sufficient to reduce the level of serum ACT. For example, the introduction of exogenous CFLs can reduce the level of serum ACT, for example, at least about 50%, 60%, 70%, 80% or 90%. The level of serum ACT may serve to indicate the degree of liver damage. Accordingly, the present invention also considers a method of reducing the degree of liver damage in a patient suffering from a shortage of CFLs, through the introduction of an effective amount of exogenous CFLs to reduce the level of serum ACT.

In some embodiments embodiment, the treatment by exogenous CFLs can reduce the level of serum ferritin. Thus, in some embodiments embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum ferritin, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared with the level before treatment. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum ferritin of at least about 50%. In another embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum ferritin of at least about 60%. In one embodiment, the treatment by exogenous CFL is to�sufficient to reduce the level of serum ferritin, at least about 70%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum ferritin of at least about 80%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum ferritin of at least about 90%. In one embodiment, the treatment by exogenous CFL is sufficient to reduce the level of serum ferritin of at least about 95%.

In one embodiment, for example in the treatment of BV and BNAH or other manifestations of deficiency of CFLs, splenomegaly weakens considerably when returning the size of the spleen to the larger than normal size by an amount of from about 1% to about 60%. The term "normal" in this context refers to the spleen of normal size in a patient who received the treatment, determined by a specialist practitioner in the field of medical science. In one embodiment, the size of the spleen is reduced to a value exceeding the normal size by an amount of from about 1% to about 50%. In another embodiment, the size of the spleen is reduced to a value exceeding the normal size by an amount of from about 1% to about 40%. In one embodiment, the size of the spleen is reduced to a value exceeding�th normal size by an amount of from about 1% to about 30%. In another embodiment, the size of the spleen is reduced to a value exceeding the normal size by an amount of from about 1% to about 20%. %. In another embodiment, the size of the spleen is reduced to a value exceeding the normal size by an amount of from about 10% to about 20%. For example, the size of the liver may be at 10%, 11%, 12% 13% 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the normal size. In yet another embodiment, the size of the spleen is reduced to a value exceeding the normal size by an amount of from about 0% to about 10%. For example, the size of the spleen may be on 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% more than the normal size of the spleen.

In one embodiment, the introduction of exogenous CFL is sufficient to reduce lymphadenopathy (i.e., enlarged lymph nodes). Thus, in some embodiments embodiment the size of the lymph nodes is reduced to a size exceeding the normal size by an amount of from about 1% to about 60%. The term "normal" in this context refers to the lymph nodes of normal size in a patient who received the treatment, determined by a specialist practitioner in the field of medical science. In one embodiment, the size of the lymph nodes is reduced to a value exceeding the normal size by an amount of from about 1% to about 50%. In another embodiment, the size �imposal down to the value that beyond normal size by an amount of from about 1% to about 40%. In one embodiment, the size of the lymph nodes is reduced to a value exceeding the normal size by an amount of from about 1% to about 30%. In another embodiment, the size of the lymph nodes is reduced to a value exceeding the normal size by an amount of from about 1% to about 20%. In another embodiment, the size of the lymph nodes is reduced to a value exceeding the normal size by an amount of from about 10% to about 20%. For example, the size of the lymph nodes can be 10%, 11%, 12% 13% 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the normal size. In yet another embodiment, the size of the lymph nodes is reduced to a value exceeding the normal size by an amount of from about 0% to about 10%. For example, the size of the lymph nodes can be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% more than the normal size of lymph nodes. In another embodiment is performed lipid analysis to monitor improvement. For example, lipid analysis can be performed to assess therapeutic effect of exogenous CFL. Lipid analysis can be performed for the tissue sample of the patient (e.g., a blood sample, a biopsy sample of the liver) by any suitable method, such as, but not limited to, liquid chromatography high resolution�Oia, gas chromatography, mass spectroscopy or thin layer chromatography or any combination of these methods, which experts in the field believe is reasonable. In one embodiment, the lipid analysis performed in accordance with the invention, shows the level of total cholesterol, triglycerides, low density lipoproteins, high density lipoproteins and/or cholesterol ester.

In one embodiment, for example, in the treatment of BV and BNAH or other manifestations of deficiency of CFLs, lipid analysis of a patient receiving treatment in accordance with the invention, shows the normalization of the concentration of lipids in the liver, spleen, intestinal tract, lymph nodes and/or the aorta, as may be determined by a specialist practitioner in the field of medical science.

The lipid level may be assessed using lipid analysis of plasma or lipid analysis tissues. If lipid analysis of plasma can be collected blood plasma, and total cholesterol in plasma can be measured using, for example, colorimetrically analysis using a set of COD-PAP (Wako Chemicals), total triglycerides in plasma can be measured using, for example, a set Triglycerides/GB (Boehringer Mannheim), and/or total cholesterol in the plasma can be determined using the set of Cholesterol/HP (Boehringer Mannheim). If lipid analysis of tissue lipids mo�ut to be extracted, for example, from the liver, spleen, and/or small samples of the intestinal tract (for example, using the method of Falsa presented in Folch et J. Biol. Chem 226:497-505 (1957)). The concentration of total cholesterol in tissue may be measured, for example, using O-phthalic aldehyde.

In some embodiments embodiment the exogenous CFL is sufficient to improve the absorption of nutrients. In one embodiment, the introduction of exogenous CFL improves the absorption of nutrients, as shown by the measured levels of serum alpha-tocopherol, 25 IT is of vitamin D, serum retinol, delegitimize or transthyretin.

In some embodiments of the incarnation, for example in the treatment of BV and BNAH or other manifestations of deficiency of CFLs, the introduction of exogenous CFL is sufficient to increase the level of serum hemoglobin (b). In one embodiment, the hemoglobin level increases by at least about 10% to about 20% compared with the level observed prior to the introduction of exogenous CFL.

In some embodiments, embodiments of exogenous CFLs may be administered using methods to minimize side effects. For example, the introduction of exogenous CFLs can minimize the immune response to exogenous CFL.

CFLs and pharmaceutical compositions comprising exogenous CFL

The present invention races�matrial treatment of any associated with deficiency of CFLs States described in this application, and other conditions, not previously mentioned, but which can be improved with treatment. Exogenous CFLs used in accordance with the invention includes recombinant CFLs, which can be produced in any expression system is useful protein, including, without limitation, cell culture (e.g., Cho cells, COS cells), bacteria, such as E. coli, transgenic animals such as mammals and birds (e.g., chicken, duck and Turkey) and the plant systems (e.g., duckweed and tobacco). One aspect of the invention relates to recombinant CFLs produced in accordance with U.S. patent No. 7524626, issued October 3, 2006; applications for U.S. patent No. 11/973853, filed October 10, 2007; No. 11/978360, filed October 29, 2007; and 12/319396, filed January 7, 2009, the disclosure of which is included in this application in its entirety by reference. One aspect of the invention relates to recombinant CFLs produced in accordance with the description in Du et al., (2005) Am. J. Hum. Genet. 77: 1061-1074, and Du and others (2008) J. LipidRes., 49: 1646-1657, the disclosure of which is included in this application in its entirety by reference. In one useful embodiment, the exogenous CFLs produced in the oviduct of transgenic birds (e.g., transgenic chicken), for example, in accordance with the method described in the application PCT/US2011/033699, filed April 23, 2011, in which�Lucena in this application in its entirety by reference. In some embodiments, embodiments of the recombinant CFLs are produced in cell lines of birds. In some embodiments, embodiments of the recombinant CFLs produced in mammalian cell lines (e.g., human).

In one embodiment, an exogenous acidic lysosomal lipase used in accordance with the invention, contains glycans with core N-acetylglucosamine (GlcNAc) and complete patterns with mannose N-bonds. GlcNAc and completed mannose glycans on exogenous CFLs can be specifically recognized and internalized by macrophages and fibroblast. Mannose-6-phosphate (MR) that can deliver proteins to the GlcNAc receptor/mannose, which are expressed on cells during conditions cure by introducing exogenous CFLs, usually there is also to exogenous CFLs used in accordance with the invention.

Usually exogenous CFL in accordance with the invention described and disclosed in this application, is a CFL man. In one embodiment, the exogenous CFL has the amino acid sequence presented in Genbank gene RefSeq NM_000235.2). In one embodiment, exogenous Mature CFL has the following amino acid sequence:

SGGKLTAVDPETNMNVSEIISYWGFPSEEYLVETEDGYILCLNRIPHGRKNHSDKGPKPVVFLQHGLLADSSNWVTNLANSSLGFILADAGFDVWMGNSRGNTWSRKHKTLSVSQDEFWAFSYDEMAKYDLPASINFILNKTGQEQVYYVGHSQGTTIGFIAFSQIPELAKRIKMFFALPVASVAFCTSPMAKLGRLPDHLIKDLFGDKEFLPQSAFLKWLGTHVCTHVILKELCGNLCFLLCGFNERNLNMSRVDVYTTHSPAGTSVQNMLHWSQAVKFQKFQAFDWGSSAKNYFHYNQSYPPTYNVKDMLVPTAVWSGGHDWLADVYDVNILLTQITNLVFH ESIPEWEHLDFIWGLDAPWRLYNKIINLMRKYQ (SEQ ID NO:1)

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

In some embodiments, embodiments of exogenous CFL is a recombinant protein CFL, as described in the application PCT/US2011/033699, filed April 23, 2011, which is incorporated into this application in its entirety by reference.

Revealed that the positions of the non-identical amino acid often differ by conservative substitution of amino acids, where amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the relative sequence identity may be adjusted upwards to correct the conservative nature of the substitution. Means of implementation of such regulation are well known to specialists in this field. Quantification conservative substitution can be performed by calculation in accordance with, for example, the algorithm of Meyer and Miller, Meyers and Millers, Computer Applic. Biol. Sci. 4:11-17 (1988).

The term "comparison window" refers to a segment of contiguous positions, such as from about 25 to about 400 positions, or from about 50 to about 200 positions, or from about 100 to about 150 positions, in which serial�activities can be compared with the reference sequence with the same number of contiguous positions after optimal alignment of two sequences. Methods of alignment of sequences for comparison are well known in this field. Optimal alignment of sequences for comparison may be performed, for example, in accordance with the algorithm of the local homology (Smith and Waterman, Adv. Appl. Math. 2:482 (1981), an algorithm for global alignment (Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for ways of similarity (Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85:2444 (1988); Altschul, etc., Nucl. Acids Res. 25:3389-402 (1997), by computerized implementation of these algorithms (e.g., GAP, BESTFIT, FASTA and BLAST in the software package Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., 30 Madison, Wis.), usually using the default settings, or by manual alignment and visual inspection (see, e.g. Current Protocols in Molecular Biology, Ausubel et al., 1994). For example, the search for protein BLAST can be performed using the XBLAST program, label=50, word length=3 to obtain amino acid sequences that have more than 80% identical to the amino acid sequence SEQ ID NO:1 or its fragment.

One example of a useful implementation of the algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignment. The program can also plot dendogram showing cluster ratio used to create the alignment. PILEUP IP�olset simplify the method of progressive alignment Feng and Doolittle, J. Mol. Evol. 35:351-360 (1987). Used a method similar to the method described by Higgins and Sharp, CABIOS 5:151-3 (1989). Procedure multiple alignment begins with the pairwise alignment of the two most similar sequences, forming a cluster of two aligned sequences. This cluster can then be combined with the next most related sequence or cluster of two aligned sequences. Two clusters of sequences can be combined by simply adding the pairwise alignment of two individual sequences. A series of such pairwise alignments, which includes sequences with increasing dissimilarity at each iteration, produces the ultimate combination.

In some embodiments embodiment the exogenous polypeptides CFL in accordance with the invention include variants of wild-type sequences. These variants fall into one or more of three classes: replacement, insertion and deletion options. These options may be an allelic or species variants are naturally occurring, or they can be prepared by site specific mutagenesis of nucleotides in the protein encoding DNA. Site-specific mutagenesis may be performed using cassette or PCR mutagenesis or other techniques well known in this� area to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Fragments of the target protein variants containing up to 100-150 amino acid residues can be prepared by in vitro synthesis, using accepted methods. Table a conservative substitution containing functionally similar amino acids are well known in this area (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U. S. A. 89:10915- 10919 (1992)).

Amino acid substitution typically performed for a single residue. Inserts are typically performed for about 1 to about 20 amino acids, although considerably larger insertions valid. The range of deletions is from about 1 to about 20 residues, although in some cases deletions may be much longer. Substitution, deletions and insertions, or any combination can be used to achieve the final derivative.

In some embodiments, embodiments of exogenous CFL has a specific activity equal to at least about 100 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity equal to at least about 200 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity equal to at least about 250 U/mg. In some embodiments, embodiments of exogenous CFL has specific activity of from example�about 100 U/mg to about 1000 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity from about 100 U/mg to about 500 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity from about 100 U/mg to about 350 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity of approximately 200 U/mg to about 350 U/mg. In some embodiments, embodiments of exogenous CFL has specific activity of from about 250 U/mg to about 350 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity, approximately 250 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity of approximately 275 U/mg. In some embodiments, embodiments of exogenous CFL has a specific activity of approximately 300 U/mg. CFL has 6 potential sites in its amino acid sequence for glycosylation with N-links: Asn36, Asn72, Asn101, Asnl61, Asn273, and Asn321, as formulated in the LAST. ID no:1. In some embodiments, embodiments at least 1, 2, 3, 4 or 5 of the sites of glycosylation with N-bonds glycosylases. In some embodiments of the incarnation all six glycosylation sites glycosylases. In some embodiments of the incarnation Asn36, Asn101, Asnl61, Asn273 and Asn321 glycosylases. In some embodiments of the incarnation Asn36, Asn101, Asn161, Asn273 and Asn321 glycosylases and Asn72 not glycosylases. In n�which variants of embodiment N-glycanova structures contain bi-, three - and tetraantennary patterns with N-acetylglucosamine(GlcNAc), mannose and/or mannose-6-phosphate (MR). In some embodiments, embodiments of exogenous CFL contains MR-modified N-glycans at Asn101, Asn161 and Asn273. In some embodiments, embodiments of exogenous CFL does not contain glycans with 0-bonds. In some embodiments, embodiments of exogenous CFLs do not contain sialic acid. In some embodiments, embodiments of exogenous CFL has a glycosylation profile, as described in the application PCT/US2011/033699, filed April 23, 2011, which is incorporated into this application in its entirety by reference.

In some embodiments of the incarnation molecular weight exogenous CFL equal to about 55 KD.

In certain embodiments of the incarnation in the treatment of the subject can be used, a nucleic acid molecule that encodes the exogenous CFLs, for example, in the vector. The doses of nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10 to 100 or more virions per dose.

In some embodiments, embodiments of the present invention, exogenous CFL is entered in accordance with the treatment method, which includes: (1) the transformation or transfer of an implantable host cell with nucleic acid, such as vector, which�expresses th CFL or active fragment, option or derivative; and (2) implanting the transformed host cell in a mammal. In some embodiments, embodiments of the invention, the implantable host is removed from a mammal, temporarily cultivated, transformed or transferred from an isolated nucleic acid encoding the exogenous CFLs, and again implanted into the same mammal from which it was extracted. The cell can be extracted from the same site where it was implanted, but not necessarily. Such variants of the incarnation is sometimes known as gene therapy ex vivo, can provide a continuous supply of exogenous polypeptides CFLs for a limited period of time.

While therapeutic protein referred to in the present invention can recombine CFLs intended for insertion in their original form, preferably a therapeutic protein as part of a pharmaceutical composition.

Thus, the invention additionally provides pharmaceutical compositions comprising an avian derived glycosylated therapeutic proteins or their pharmaceutically acceptable derivatives together with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients and methods of administration of such pharmaceutical compositions. The invent�tion also provides pharmaceutical compositions, contains derived glycosylated therapeutic mammalian proteins, or their pharmaceutically acceptable derivatives together with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients and methods of administration of such pharmaceutical compositions.

Media (media) must be "acceptable (acceptable)" in the sense of compatibility with other ingredients of the composition and not to be harmful to recipients. Methods of treating a patient (e.g., the amount of administered therapeutic protein, frequency of administration and duration of treatment) with the use of pharmaceutical compositions in accordance with the invention can be determined using standard methodology, known to specialists in this field.

Pharmaceutical compositions include compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral administration. Pharmaceutical compositions include compositions suitable for administration by injection, including intramuscular, subcutaneous and intravenous. The pharmaceutical compositions also include compositions for administration by inhalation or insufflation. The compositions can be, where appropriate, conveniently presented in discrete units dozy�hardware and can be cooked in any way, well known in the field of pharmacy. Methods of manufacturing pharmaceutical compositions typically include the stage of compounds of therapeutic proteins with liquid carriers or finally divided solid carriers or both and then, if necessary, shaping the product into the required shape.

Pharmaceutical formulations suitable for oral administration may be conveniently presented in discrete units such as capsules, cachets or tablets each containing a predetermined amount of active ingredient in the form of a powder or granules; solution; suspension; or emulsion. The active ingredient may also be presented in the form of a ball, electuary or paste. Tablets and capsules for oral administration may contain traditional shaping, such as binders, fillers, lubricants, dezintegriruetsja substances or wetting agent. Tablets may be coated according to methods well known in this field. 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 mixing with water or other suitable medium before use. Such liquid preparations may contain traditional supplements such taxespantyhose substances emulsifiers, anhydrous environment (which may include edible oils), or preservatives.

Therapeutic proteins in accordance with the invention can also be designed for parenteral administration (e.g. by injection, for example rapid injection or continuous infusion) and may be presented in the form of doses in ampules, pre-filled syringes, containers for small volume injections or packages containing multiple doses, with added preservative. Therapeutic proteins can be introduced through, for example, subcutaneous injection, intramuscular injection and intravenous infusion or injection. In one embodiment, the exogenous CFL is introduced by means of intravenous infusion with any suitable method. In one example, exogenous CFLs may be administered by intravenous infusion through peripheral line. In another example, exogenous CFLs may be administered by intravenous infusion through a peripheral inserted Central catheter. In another example, exogenous CFLs may be administered by intravenous infusion using machines for outpatient infusion connected to the vascular access port. In one embodiment, intravenous infusion of the drug is injected during the time period from 1 to 8 hours, depending on the�STI from the number of medicines intended for infusion, and the history of the patient's reactions during previous infusions, as determined by a doctor or specialist in this area. In another embodiment, the exogenous CFL is injected through intravenous injection. In another embodiment, the exogenous CFLs may be administered by intraperitoneal injection. In yet another embodiment, the exogenous CFL is introduced by means of a pharmaceutically acceptable capsules therapeutic protein. For example, the capsule may be a gelatin capsule with an enteric shell.

In some embodiments embodiment therapeutic proteins are introduced by means of infusion, and the infusion may be carried out during an extended period of time, for example, from 30 minutes to 10 hours. Thus, the infusion can be carried out, for example, within a time period of about 1 hour, about 2 hours, about 3 hours, about 4 hours or about 5 hours. The infusion can also be carried out at different speeds. For example, the rate of infusion may be equal to from about 1 ml per hour to about 20 ml per hour. In some embodiments embodiment the rate of infusion is equal to from about 5 ml per hour to about 10 ml per hour. In one embodiment, the rate of infusion 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. In one embodiment, the speed vli�number equal to from 0.1 to 5 mg/kg/h. In one embodiment, the rate of infusion 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/h.

Therapeutic proteins can have such forms as suspensions, solutions or emulsions in oily or aqueous media, and can include forming means as suspendida, stabilizing and/or dispersing agents. Therapeutic proteins can be used in powder form, obtained by aseptic separation of sterile solid or by lyophilization from solution, for mixing with a suitable medium, such as a sterile non-pyrogenic water, before use.

For topical administration to the epidermis therapeutic proteins can be in the form of ointments, creams or lotions, or transdermal patches. Ointments and creams may, for example, be aqueous or oily base with the addition of suitable thickeners and/or gelling agents. Lotions can be water or oil based and usually also contain one or more emulsifiers, stabilizers, dispersants, suspendida agents, thickeners or dyes.

Formulations suitable for topical administration in the mouth include tablets containing the active ingredient with a flavored basis, usually sucrose and acacia or tragakant; tablets containing the active in�radiant with inert basis, such as gelatin and glycerin or sucrose and acacia; and liquid mouth rinse containing the active ingredient with a suitable liquid carrier. Pharmaceutical formulations suitable for rectal administration, where the carrier is a solid material, most preferably have the form of suppositories with a single dose. Suitable carriers include cocoa butter and other products commonly used in this area, and suppositories should easily be made from a mixture of the active compound with the softened or melted carrier (carriers) followed by chilling and shaping in molds.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing additive to the active ingredient, carriers known in the relevant field.

Intranasal therapeutic proteins in accordance with the invention can be used in the form of a liquid spray or dispersible powder or in the form of drops.

Drops can be formed from aqueous or nonaqueous basis, and also comprising one or more dispersing agents, solvents or suspendida substances. Liquid sprays are fed from cylinders under pressure.

For inhalation administration of therapeutic proteins in accordance with the invention can simply be delivered � of insufflator, nebulizer or pressurized vessel or other suitable means of supplying an aerosol. Pressurised containers may include a suitable gas propellant, such as DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of an aerosol under pressure, the unit dose can be set by application of the valve for feeding the necessary amount.

For inhalation or insufflation administration, therapeutic proteins in accordance with the invention may take the form of a dry powder composition, for example a powder mixture of the composition and the corresponding powder base, such as lactose or starch. The powder composition may be presented in the form of unit doses, for example, capsules or cartridges or e.g. gelatin or blister packs from which the powder may be entered using the inhaler or insufflator. When required, the above-described compositions intended for continuous release of the active ingredient, can be used.

Pharmaceutical compositions in accordance with the invention can also contain other active ingredients such as antimicrobials or preservatives.

In some embodiments embodiment, the concentration of exogenous CFLs in the pharmaceutical composition is from when�Erno 0.5 to about 10 mg/ml. In some embodiments embodiment, the concentration of CFL is equal to from about 1 to about 5 mg/ml In some embodiments embodiment, the concentration of CFLs equal to 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.

In some embodiments, embodiments of the pharmaceutical composition comprising exogenous CFL, further comprises a buffer. Typical buffers include acetate, phosphate, citrate, and glutamate buffers. Typical buffers include citrate of lithium, sodium citrate, potassium citrate, calcium citrate, lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, malate sodium malate potassium malate calcium, lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate and mixtures thereof. In some embodiments embodiment the buffer is a dihydrate of trisodium citrate. In some embodiments embodiment the buffer is a citric acid monohydrate. In some embodiments embodiment the pharmaceutical composition comprises a trisodium citrate dihydrate and citric acid monohydrate.

In some embodiments, vopl�cluding pharmaceutical composition, including exogenous CFL, further comprises a stabilizer. Typical stabilizers include albumin, trehalose, sugars, amino acids, polyols, cyclodextrin, salts such as sodium chloride, magnesium chloride and calcium chloride, bioprotector and mixtures thereof. In some embodiments embodiment the pharmaceutical composition comprises human serum albumin.

In a specific example, recombinant CFLs person made in accordance with the description in this application, is used in the pharmaceutical composition, where each 1 milliliter contains exogenous CFLs (e.g., 2 mg CFLs), trisodium citrate dehydrate (e.g., about 13.7 mg), citric acid monohydrate (for example, 1,57 mg) and human serum albumin (e.g., 10 mg) and is given to acid number pH 5,9±0,1. The present invention considers any route of administration that facilitates the entry of exogenous CFLs in lysosomes of the respective organs or tissues.

EXAMPLES

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

Example 1

Treatment of deficiency of CFLs with early onset (Wolman disease) through the introduction of recombinant CFL.

A boy at 15 weeks of age was sent to the hospital due to low weight gain birth day (weight �ri birth 3,88 kg). The patient was admitted with vomiting, eating problems, poor nutritional status, diarrhea, increased bloating and anemia. Diagnosed with Wolman disease.

Upon initial physical examination the weight of the patient amounted to 5.62 kg, which is equivalent to 5% on a scale depending on the weight of age. During the following 4 weeks after the initial examination at 15 weeks of age and before the initial infusion at 19 weeks of age the weight of the patient is not increased. The estimated growth rate was less than 1 per cent on a scale depending on the weight of age. Belly was visibly swollen due to a significant hepatomegaly and splenomegaly. Ultrasound examination and CT scan confirmed hepatosplenomegaly and bilateral symmetric increase of the adrenal gland with calcification. The level of serum alanine aminotransferase (ALT) was 119 U/l (normal level is 10-50 U/l), and serum levels of aspartate aminotransferase - 216 E/l) (normal level - 10-45 U/l). Before treatment, the number of serum ferritin, an indicator of inflammation, was approximately 1500 µg/l (normal level - 7-144 µg/l). Before treatment there was a steady anemia when the hemoglobin level is from 7.2 to 8.3 g/DL.

At 19 weeks of age were initiated weekly in�otrivine infusion rhLAL (SBC-102) with an initial dose of 0.2 mg/kg. Was no prior preparation of the patient with the introduction of 1 mg/kg diphenhydramine approximately 90 minutes before the infusion of SBC-102 for prevention of possible reactions to the infusion. The duration of infusion was approximately 4 hours. Injections were correctly calculated, and the patient had no adverse events or reactions.

The second infusion was performed to the patient within seven days after the first infusion. The patient was introduced dose, equal to 0.3 mg/kg, SBC-102, for about 4 hours without signs of adverse events.

Within two weeks after the start of treatment, the patient had marked improvement of General well-being, including increased vitality and receptivity. Diarrhoea and vomiting were stable. The patient began to gain weight and showed a marked decrease in the number of serum transaminases (e.g., AST and ALT) are mostly back to normal (Figures 1A and 1B). The rate of growth of the patient rapidly returned to normal (Figures 3 and 4). Bloating decreased correspondingly to the reduction of stomach size. Tests of liver function showed continuous improvement (Figures 1A and 1B).

At the third visit, the patient received a dose of SBC-102 0.5 mg/kg. Infusion was performed without adverse events. Clinical condition showed continuous improvement with led�increase the weight by 150 g after 7 days and increase the circumference of the shoulder to 1.5 cm after the start of treatment (Figures 3 and 4). The liver tests were stable, the hemoglobin level increased (10-11 g/DL), and ferritin levels continued to decline (Figure 2). Alkaline phosphatase levels before treatment was normal and was 137 U/l (normal level - 110-300 (E/l) and increased in the treatment (204 U/l). The effect of the introduction of SBC-102 was stable and was confirmed by the observations for the pre-clinical models of disease.

Beginning with the fourth infusion, the patient began to receive a weekly dose of 1.0 mg/kg in two months after the start of treatment process, patients are mostly improved at an estimated growth rate of about 95%. This improvement in growth led to increase the weight by 1.25 kg 2,79 or pounds in 63 days if the weight 7,21 kg, which corresponded to 30% on a scale depending on the weight of age (Figures 3 and 4). Levels ACT and ALT levels decreased rapidly after the first infusion.

After three months of treatment, the levels of ACT and ALT became normal. In addition to improving the liver function was observed a significant decrease in ferritin (Figure 2).

After 4-month treatment symptoms in the gastrointestinal system of the patient was not seen and the nutritional status of the patient was excellent. The patient continued to gain weight (Figures 3 and 4) and showed physical signs of a normal healthy child. The patient continued normally carry infusion without any reactions or side �of effektov. The patient received the 21st dose of 1.0 mg/kg as an ambulatory patient.

Example 2

Design clinical studies deficiency CFL with early SBC-102, is a recombinant CFLs produced in transgenic rooster, is introduced through weekly intravenous infusion. The study is designed to evaluate the safety, tolerability and efficacy of two dosing schedules of SBC-102 at weekly intravenous infusion. However, the main output variables in this study to determine the safety and tolerability of SBC-102 children with impaired growth due to a deficiency of CFLs and include: main features of the state and results of physical examination; clinical laboratory tests; tests for antibodies to the medication and the use of appropriate medicines. Given that the growth impairment in children is a universal clinical sign of deficiency CFL/Wolman phenotype, successful treatment of this disease should affect the growth impairment observed in children suffering from deficiency of CFLs. The parameters related directly to the child's growth and nutritional status, assessed as a secondary or diagnostic purposes: for example, the rate of incremental growth related to weight; gain weight; and the rate of linear growth. This study also examines the impact of SBC-102 at �farmakodinamika biomarkers; the size of the liver and spleen, lymphadenopathy; hemoglobin and platelets; laboratory assessment of liver function and nutrition; the size of the stomach, srednevasanta arm circumference; and head circumference. This study also describes the preliminary pharmacokinetics of SBC-102 in children with growth disorders due to a deficiency of CFLs, including the Cmax for plasma and estimated clearance.

tr>
Table 1
Schedule of assessments: observation for 24 weeks
AssessmentNach.diagnosisWeeks. 1Weeks.2Weeks.3Weeks.4Weeks. 6Weeks.8Weeks. 10Weeks. 12Weeks. 14Weeks. 16Weeks. 18Weeks. 20Weeks. 22Weeks. 24
The pre. infusion±2 days±2 days±2 days±2 days±2 days ±2 days±2 days±2 days±2 days±2 days±2 days±2 days±2 days±2 days
Informed consentX
The inclusion / exclusionX
HistoryX
Sample for molecular genetic analysis
The method of "dry drop"
Therapeutic-XXX
health
12-lead ECGXXp
Physical examination2XXpXp
Gest pregnancy3XXpXpXpXpXp
Clinical laboratoryXXp XpXp
Liver, lipidXXpXpXpXp
Acute phaseXXpXpXpXp
Anti SBC-102 XXpXpXpXpXpXp
The collection of biomarkers in serum and urineXXpXpXpXpXpXp
MRI/MRS6bellyXX
The main features of7XXXXXXXXX9XXXXX
The infusion of SBC-102XXXXXXXXX10XXXXX
Adverse eventsContinuously
The appropriate medication/treatmentContinuously
pPre-infusion.
1Quality of life caused by the condition in line with age.
2The physical examination will include measurement of weight (growth only at survey), estimating the size of the liver and spleen, lymphadenopathy and arterial disease.

The subjects are recorded in two consecutive groups of the same size (4 subjects in each group). The dosage varies within each group and between groups, since the introduction of the doses first subject in the group with lower doses (Group 1; the initial dose of 0.35 mg·kg-1). The introduction of additional doses of the subjects in Group 1 and the beginning of doses in the group with higher doses (Group 2; initial dose 1 mg·kg-1based on acceptable safety and tolerability of the previous entities.

Group 1

The first four subjects registered in the study constitute a Group 1. The first entity in the group receives a single dose of 0.35 mg·kg-1SBC-102 and if approved the continuation of doses based on the analysis of security within at least 24 hours after the dose, receives a second dose of 0.35 mg·kg-1SBC-102 in one week. After administration to the subject a second dose of SBC-102, all available data relating to security, �analiziruyutsya and the decision regarding admissibility i.e. opportunities, increasing doses first subject to 1 mg·kg-1and the beginning of the dose to other entities in Group 1. The dose of the other three subjects from Group 1 is identical. If the safety analysis cannot guarantee the safety of increasing doses, administered to a subject, with 0.35 mg·kg-1to 1 mg·kg-1but shows the safety further treatment of the subject with the introduction of the initial dose, the subject may continue to receive a dose of 0.35 mg·kg-1. If any entity in the Group 1 exhibits a suboptimal treatment response after receiving at least 4 doses of 1 mg·kg-1SBC-102, is considered an additional increase dose to 3 mg·kg-1.

Group 2

The beginning of doses in Group 2 is carried out after full registration Group 1 and analysis of a security for at least 2 subjects who received 2 or more doses of 1 mg·kg-1SBC-102 in Group 1. The remaining 4 subjects involved in the study and register and get a dose in Group 2. The first subject in this group receives a single dose of 1 mg·kg-1SBC-102 and if approved the continuation of doses based on the analysis of security within at least 24 hours after the dose, receives a second dose of 1 mg·kg-1SBC-102 in one week. After administration to the subject a second dose of SBC-102 all access�s data regarding safety, analyzed, and a decision on admissibility, i.e. the possibilities of increasing doses first subject to 3 mg·kg-1and the beginning of the dose to other entities in the Group 2. The dose of the other three subjects from Group 2 is identical, with the safety analysis. If the safety analysis does not guarantee the safety of increasing doses, administered to the subject, 1 mg·kg-1to 3 mg·kg-1but shows the safety further treatment of the subject with the introduction of the initial dose, the subject may continue to receive a dose of 1 mg·kg-1. If the initial dose of 1 mg·kg-1not well tolerated by the subject, may be considered the introduction of a low dose of 0.35 mg·kg-1.

The study consists of approximately 22 visits in accordance with the schedule: Visit 1 (inspection), Visit 2 (baseline assessment, the beginning of the use of study medication) at Visit 21 (weekly administration of the investigational medicinal product), the Visit 22 (completed end of study). Given the complexity of life and the importance of treatment of impaired growth with an early start due to a deficiency of CFLs, it is likely that these subjects will be hospitalized.

Research will involve boys and girls with impaired growth due to a deficiency of CFLs. Actors teaching�experiencing in research if all of the following criteria: (1) the parent or legal guardian fully understands the nature and purpose of the research including possible risks and side effects, and gives informed consent/permission before performing any research procedures; (2) a boy or a girl with low activity of CFLs relative to normal levels, documented by the laboratory performing the analysis, or the documented results of molecular genetic studies, confirming the diagnosis of deficiency of CFLs; and (3) breach growth with early onset at age less than 6 months.

Security

The main points of security include the occurrence of adverse events (AES) and reactions related to the infusion (DOM);

deviations from the basis of the fixed signs (blood pressure, heart rate, respiratory rate, temperature), results of physical examinations and clinical laboratory tests (CBC/Hematology, serum chemistry and urine analysis); use of appropriate medications/treatments; and characterization of antibodies to SBC-102 (ADA), including the conversion rate of serum, time to conversion of serum, the titer of ADA average and peak immunoglobulin G (IgG) and the time before the ADA titer IgG peak.

Efficiency

The main points of performance include: (1) deviation and/or the relative deviation from the baseline size of the liver and spleen by ultrasonic�audio research) and the volume of the liver and spleen, and fat (via magnetic resonance image [MRI]); and (2) deviation from baseline serum transaminase, serum lipids (total cholesterol, triglycerides, high-density lipoprotein [HDL] and low-density lipoprotein [LDL]), level of hemoglobin and platelets. Growth parameters, including deviation from baseline in percentage and z-scores are also estimated for subjects younger than 18 years of age. These growth parameters based on the maps of the growth Centers of disease control (CCB) and include maps of the dependence of weight on age (CC), weight growth (BP), height age (PB) and head circumference by age (GIA) among subjects whose age is less than 30 months, and BB, physique age (TV; the relation of physique to growth) and weight of body (W) in subjects whose age is greater than or equal to 36 months and up to 18 years of age, and also with a corresponding increase indicators of underweight, wasting and short stature in all subjects.

Example 3

Physical assessment of patients with deficiency of CFLs with early onset

Patients clinically stable enough to transfer General anesthesia, should be considered from the point of view of possibility of use of the Central catheter for long-term access to the vascular system. The subjects exposed to General anesthesia and/or effects of sedation for other procedures considered basic magneto�esonance image (MRI) of the abdomen. In the case of new procedures requiring General anesthesia and/or effect of sedatives, is considered a control MRI, if it's been at least three months after the first infusion. Measured anthropometric characteristics (weight, height, abdominal circumference, srednevasanta arm circumference and head circumference). Performed General physical examination. A full physical examination. The examination includes evaluation of General view of the subject, skin, head, eyes, ears, nose and throat, heart, lungs, abdomen, extremities/joints and neurological conditions. Every physical examination also includes the following:

The size of the liver: is clinical estimation of liver size (palpable/reprodutiva and centimeters below costal edge), correctness (smooth/nodular) and sensitivity (painful/not painful).

The size of the spleen: performs clinical assessment of the size of the spleen (palpable/reprodutiva and centimeters below costal edge), correctness (smooth/nodular) and sensitivity (painful/not painful).

Lymphadenopathy: evaluated the size, location and nature of all palpable lymph nodes. Intended for survey areas include: headache (occipital, situated forward of the ear, located behind the ear �akawini, submental, submandibular), cervical, clavicular, axillary and inguinale. All enlarged nodes are characterized as painful and not painful.

Photo: is a digital image of the subject in the supine position (full height and magnified image of the abdomen).

Ultrasound examination and MRI of the liver/spleen

Ultrasonography of the abdomen can be performed to measure the size of the liver and spleen. MRI of the abdomen can provide a better quantification of the volume of the liver and spleen and can be considered at baseline and at the visit after at least 3 months after the first infusion.

Main features

The main signs include pulse rate, respiratory rate, systolic and diastolic arterial pressure, and Central body temperature (rectal or oral). Assessment of heart rate and blood pressure is performed after the position of the subject lying on his back. The main signs are measured at all visits during the study. In the days of doses the main symptoms recorded prior to infusion, every 15 minutes (±10) during the infusion and for 2 hours after infusion, and then every 30 minutes (±15) in the period from 2 to 4 hours after completion of infusion.

Example 4

Laboratory evaluation

The following laboratory� assessments are carried out in the form of diagnostic tests and to determine the effectiveness of:

1) General analysis of blood/Hematology: leukocyte count in the blood, the level of red blood cells, hemoglobin, hematocrit, average cell volume (RMSE), mean erythrocyte hemoglobin (SAG), srednegalechny concentration of hemoglobin (SCCG), the level of platelets, neutrophil, lymphocytes, monocytes, eosinophils, basophils, peripheral blood smear for the study of cell morphology.

2) blood chemistry: glucose, bun, creatinine, sodium, potassium, chloride, calcium (total and ionized), magnesium, inorganic phosphorus, total protein, lactate dehydrogenase.

3) liver function Tests: ACT/serum aspartate aminotransferase (SAST), ALT/serum of alanineaminotransferase (SALT), alkaline phosphatase, gammaglutamyltransferase (GGT), albumin, bilirubin (direct, total)

4) antibodies to the drug: antibody to SBC-102

5) urine Analysis: pH, glucose, ketones, blood, protein, nitrite

6) Study of coagulation: leukocytes (can be made microscopic examination, if there are deviations from the norm for blood, nitrite and/or leukocytes)

7) Laboratory evaluation of food: alpha-tocopherol serum:

the ratio of cholesterol, 25 HE vitamin D, serum retinol, delegitimation, transthyretin, serum ferritin

8) lipid profile: total cholesterol, triglyceride, HDL, IL�

9) Genetic profile

DNA sequences, including the sequence encoding the protein and the sequences that regulate gene transcription, the stability of information ribonucleic acid (mRNA) and efficiency of protein translation, which can be identified include:

1. Acidic lysosomal lipase gene (LIPA)

2. The genes encoding for other proteins involved in lipid biology that may contribute and/or to modify the phenotype of the deficiency of CFLs, for example ASA

3. Genes that can modify susceptibility to any SBC-102

10) Pharmacokinetic evaluation

To reduce the risk of iatrogenic anemia FC the sample may be limited. In order of importance, samples are collected to determine the following parameters: 1) Cmax and 2) evaluation of lethal concentration (LC). Collected samples for measurement of serum levels of SBC-102 in day (1 dose) and day 105 (dose 16). All players in samples taken before the introduction of dose (within 30 minutes of dose); 90(±5) minutes after the start of infusion; and through 110(±5) minutes after the start of infusion. All FC samples at time points coinciding with the evaluation of key attributes should be taken to inflate the cuff to assess blood pressure (CD) on his arm where the injection is not performed. FC samples at other time points are taken, less� least 5 minutes after deflation of the cuff.

Example 5

Preparation of dose and infusion

SBC-102 is available in glass vials with single dose of 10 ml in the form of a transparent liquid. The solution (10.5 ml, including 5% overflow) has a concentration of 2 mg·ml-1. All tubes with SBC-102 was kept at a controlled temperature of 2-8°C. the Tubes are frozen and protected from light during storage. The syringe containing the drug SBC-102, diluted in 0.9% salt solution was prepared immediately before injection. After preparing the syringe with SBC-102 diluted solution was labeled and used within 4 hours after preparation.

The weight of the patient, recorded before administration of the morning dose on the day of infusion and rounded to the nearest 0.1 kg was used to calculate the volume of SBC-102 for each infusion. Total injections used in the study, based on the mode of doses, shown in Table 2.

Preparation and introduction of the dose should be performed using sterile non-pyrogenic disposable materials, including, but not limited to, syringes, needles, transition tube and burette with stopcock.

The rate of infusion must be installed on the flow controller in such a way as to introduce the entire amount within about 120 minutes, as shown in Table 3.

Table 2
DoseThe volume of infusion
0.35 mg/kg10 ml
1 mg/kg10 ml
3 mg/kg20 ml
Table 3
DoseA flow per hourA flow per minuteConsumption per kilogram per hour
0.35 mg/kg5 ml0,083 ml0,175 mg/kg/h
1 mg/kg5 ml0,083 ml0.5 mg/kg/h
3 mg/kg10 ml0,167 ml1.5 mg/kg/h

Example 6

Adverse events (AE)

In the case of observation of subjects NYA or reactions related to the infusion (the MOAT) with clinically significant cardiovascular, respiratory or other effects, the infusion should be beauti�eno and the subject should receive treatment to correct anaphylactically reaction in accordance with the guidelines for the management of severe reactions at the infusion in children whose age is less than 2 years. Treatment may include intravenous anthistamines, corticosteroids and epinephrine if needed. For related biological products the majority of detainees DITCH appears later than 24 hours after the infusion. Symptoms include arthralgia, myalgia, symptoms similar to the symptoms of the flu, a headache, fatigue and irritation or hives. Delayed reactions can be treated with analgesics or antihistamines in accordance with clinical indications. The DITCH is classified as acute or (manifesting within 24 hours after the start of infusion), or as delayed (manifested within 1 to 6 days after injection). Medicines and equipment for the treatment of hypersensitivity reactions should be ready for immediate use in case of unexpected adverse hypersensitivity reactions. These tools include, but are not limited to, oxygen, acetaminophen, antihistamines (eg, diphenhydramine, parenteral and oral drugs), corticosteroids, epinephrine and equipment for reconstruction of cardiac activity and respiration. In such biological products are the most acute MOAT occur within 24 hours of infusion (instructions for medical use of the drug is Cerezyme®, VPRIV®, Fabrazyme®). Signs of possible acute MOAT mo�ut be classified as follows: hyperemia, a rush of blood, fever and/or chills, nausea, itching, urticaria, gastrointestinal symptoms (vomiting, diarrhea, abdominal cramps). Weak reactions are defined as self-limiting, spontaneously disappearing after a temporary reaction is complete, or reduce the rate of infusion. Secondary reactions are defined as unrecoverable by simple measures and require enhanced monitoring and the suspension of treatment. Serious reactions involve chest pain, shortness of breath, wheezing, stridor hypotension or hypertension, respiratory arrest, apnea shortness of breath, bradycardia or tachycardia. If any of the above signs or symptoms is observed during the infusion, and the subject remains hemodynamically stable, the rate of infusion may be reduced (reduction of about half the speed used in the beginning of the reaction, for example, 10 ml·h-1to 5 ml·h-1), and the infusion time was increased. After elimination of the reaction, the infusion should continue for at least 30 minutes at low speed before increasing the speed to 75% of the initial rate of infusion. If the subject continues to show signs of hypersensitivity, dose of antihistamine may be administered intramuscularly or slowly intravenously, in accordance with the guidelines for the management of severe reactions at the infusion in children whose age is less than 2 years.

Example 7

Introduced�e recombinant CLL patient-a person with deficiency of CFLs with late onset

The main purpose of the study is to evaluate the safety and tolerability of SBC-102 in patients with liver dysfunction due to a deficiency of CFLs with late onset (the main symptoms, physical examination, clinical laboratory tests, tests for immunogenicity, assessment of adverse events, appropriate treatment). A secondary objective is to characterize pharmakinetic SBC - 102, is delivered by intravenous infusion, after administration of single and multiple doses (before and after infusion, day 0 and 21). Criteria for inclusion in the composition of subjects with deficiency of CFLs with late onset following.

1. The patient fully understands the nature and purpose of the study, including possible risks and side effects, and is willing and able to comply with all procedures of the study and provides informed consent;

2. Men and women aged ≥ 18 and ≤ 65 years;

3. Decreased activity of the CFL, documented by the laboratory performing the analysis, or the documented results of molecular genetic studies, confirming the diagnosis of deficiency of CFLs;

4. Evidence of liver damage on the basis of clinical presentation (hepatomegaly) and/or the results of laboratory tests (ALT or ACT ≥ 1.5 x ULN (upper limit of normal);

5. When taking statin or ezetimibe the patient should�n to receive a stable dose, at least 4 weeks prior to the survey;

6. All women must have a negative result serum pregnancy test during the examination and cannot breastfeed; and

7. Women with reproductive potential must agree to adopt the highly effective and approved contraceptive methods throughout the study and for 30 days after the last dose.

Clinical assessment includes physical examination, urinalysis, clinical biochemical analysis of blood/Hematology; agents acute phase, the study of coagulation, 12-lead ECG, antibodies to SBC-102 and FC to manage Cmax, AUCinf, T1/2, Cl and Vss.

Patients entered the dose of 0.35 mg·kg-1, 1 mg·kg-1or 3 mg·kg-1CFL weekly by intravenous infusion for 2 hours. The first subject of the injected dose, and it is controlled by the tolerance for at least 24 hours before administration of doses to other entities of the group. Each subject remains stationary patient during 24 hours after the first infusions of SBC-102. The subjects continue to be an additional 3 doses of SBC-102 weekly intravenous, provided the tolerability and safety remain acceptable.

Pharmacokinetics

FC data is analyzed from all subjects included in the study, Paul�waiting, at least one dose of study medication, excluding any data that may have affected the deviation from the basic Protocol. FC analyses are performed using the same model infusion. Following FC parameters are determined and presented to the group (Cmax, AUCinf, T1/2, Cl and Vss) - FC parameters of single and multiple doses were compared using data from Visit 2 and Visit 6.

The proposed increase in dose in this study can be 3, 4, 5 or 6-fold, which may allow to evaluate the initial safety, tolerability and pharmacokinetic parameters in humans in a 6-fold range of doses on the basis of units of measure mg·kg-1. In relevant preclinical models using rats, pharmacodynamic effects when administered doses of 1 mg·kg-11 times weekly, 3 mg·kg-1every second week, and 5 mg·kg-1once weekly compared. Thus, although it is not expected that doses above 3 mg·kg-1once weekly required dose above 3 mg·kg-1such as 4, 5, 6, 7, 8, 9 or 10 mg·kg-1may be considered depending on the severity of the disease.

Design research

Assuming the rarity of patients with this condition are the final number for this study is 9 the subjects to be measured. The subjects included in t�and consistent groups of 3 subject in each group. The subjects included in group 1, receiving the first dose, after them - the subjects of group 2, then group 3.

X
Table 4
Schedule research deficit CFL with late onset: visits, evaluation and intervals
AssessmentThe pre. biscuitsThe active phasePostactional phase
Visit 1Visit 2Visit 3Visit 4HSVisit 5HSVisit 6HSVisit 7Visit 7.1Visit 8
(Day -(Day(Day(Day(Day(Day(Day(Day(Day(Day(Day28-7)0)1)7±1)8)14±1)15)21±1)22)28±1)35±1)52±1)
Formed consentX
Criteria for inclusion / exclusionXX
Demographic informationX
The results of the survey of the health of the patientX
History1X
12-lead ECGX X
Physical examinationXXXXpX
Key indicators3XXpXXpXpXpXX
A urine testXXpXpXXXX X
The pregnancy test4XXpXpX
HOW/HematologyXXpXpXpXpXpXX
Biochemical analysis of bloodXXpXpXpXpXpXX
Pokazateliami XXpXpXpXpXpXXX
Lipid indicesXXpX
The acute phase reactantsXXpXX
Gesta coagulationX X
Viral hepatitisX
Autoimmune hepatitisX
A DNA sampleX
Activity KJIJ ciavXXpX
Antibody to SBC-102 (ADA)XXpXX
SampleXXpXXX
research biomarker
FC sample5XX
Dosing SBC-102XXXX
Adverse eventsXXXXXXXXXXX
Appropriate treatmentXXXXXXXXXXXX
TC = a phone Call
pPreliminary dose
1Including alcoholic history (AUDIT questionnaire)
2Including height and weight
3Pre-dose, every 15 minutes during the infusion, every 15 minutes for 2 hours after the infusion and every 30 minutes during the time period from 2 to 4 hours after infusion
4Serum at visits 1 and 8; urine at visit 2 and visit 6
5Preliminary dose, 10, 15, 20, 40, 60, 90 minutes during the infusion, at the end of the infusion (approximately 120 minutes) and via 5, 10, 20, 30, 40, 60 and 120 minutes after infusion

Group 1

Three subjects will receive an intravenous dose of 0.35 mg·kg-1SBC-102. The first subject receives a dose and monitored for tolerability for at least 24hours before the introduction of dose 2 others in the group. Each subject remains stationary patient during 24 hours after the first infusions of SBC-102. Subjects administered intravenously 3 additional doses of 0.35 mg·kg-1subject acceptability tolerability and safety.

Group 2

Three subjects will receive an intravenous dose of 1 mg kg-1SBC-102. The first subject from group 2 gets the dose and monitored for tolerability for at least 24 hours before administration of dose 2 others in the group. Each subject remains stationary patient during 24 hours after the first infusions of SBC-102. Subjects administered intravenously 3 additional doses of 1 mg·kg-1subject acceptability tolerability and safety.

Group 3

Three subjects produced by intravenous infusion of a dose of 3 mg·kg-1SBC-102. The first subject in Group 3 receives a dose and monitored for tolerability for at least 24 hours before administration of the dose to other entities in the group. Each subject remains stationary patient during 24 hours after the first infusions of SBC-102. Subjects administered intravenously 3 additional doses of 3 mg·kg-1once a week, provided the acceptability tolerability and safety.

Safety Committee (KB) can stop the dose for the entire group or for individual subject at any time of vsledstvie� poor tolerability or potential risks.

If the subject of the research is interrupted treatment during the visit according to the schedule that is different from visit 8 (end of study), or during an unplanned visit, the subject will be refundable 7 days after the last dose of SBC-102 to complete research assessments during the visit 8.

SBC-102 is introduced by means of intravenous infusion during visits 2, 4, 5 and 6. Appropriate treatment are recorded throughout the study. Adverse events are recorded, starting from the moment of signing the informed consent.

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

The duration of the study

The study includes 4 weeks of doses SBC-102 and the period of cleaning to maintain the safety assessment and the schedule of doses for subsequent clinical studies. After completion of the study, the subjects may be entitled to continue to receive SBC-102 in accordance with a separate Protocol to evaluate the long-term safety and efficacy of SBC-102 in patients with deficiency of CFLs/phenotype BIH.

Physical examination

General physical examination is performed by a qualified medical specialist. Data systems (including, but �e limited to cardiovascular, respiratory, gastrointestinal and neurological system) must be specified and recorded. All deviations from the norm must be established for each execution of the survey. A new diagnosis of abnormalities should be recorded in the form of adverse events, if applicable.

Additional assessment physical examination should be performed during the screening visit:

a) the Size of the liver: is clinical estimation of liver size (palpable/reprodutiva and centimeters below costal edge), correctness (smooth/nodular) and sensitivity (painful/not painful).

b) Lymphadenopathy: evaluated the size, location and nature of all palpable lymph nodes. Intended for survey areas include: headache (occipital, situated forward of the ear, located behind the auricle, submental, submandibular), cervical, clavicular, axillary and inguinale. All enlarged nodes are characterized as painful and not painful.

b) Arterial disease: the pulse of the right and left posterior tibial artery and dorsal artery of the foot are evaluated clinically and ankle-brachial index (ABI) for the right and left side are recorded. ABI is defined as the ratio of systolic pressure in the back and�the criteria of the foot or posterior tibial artery to the systolic pressure in the right or left shoulder (whichever is higher).

Main features

The main features are measured, including heart rate, respiratory rate, systolic and diastolic blood pressure, and temperature. Assessment of heart rate and blood pressure is performed after the subject was in the supine position for at least 5 minutes. In the days of doses the main symptoms recorded prior to infusion, every 15 minutes (±5) during the infusion and for 2 hours after the infusion and then every 30 minutes (±10) in the period from 2 to 4 hours after completion of infusion. Additional readings may be used at the discretion of the researcher in the case of reactions related to the infusion (ROS). Electrocardiogram (ECG) 12-lead with formal records can be obtained after the subject was in the supine position for at least 5 minutes.

Laboratory evaluation

Samples for laboratory tests are collected at the time points specified in the schedule of assessments. The following laboratory tests (except ESR, coagulation studies and antibodies to SBC-102).

Common blood test/Hematology: leukocyte count in the blood, the level of red blood cells, hemoglobin, hematocrit, average cell volume (RMSE), mean erythrocyte hemoglobin (SAG), srednegalechny concentration of hemoglobin (SCCG), the level of platelets, neutrophil, lymphocytes, m�nocity, eosinophils, basophils.

Blood chemistry: glucose, bun, creatinine, sodium, potassium, chloride, calcium, magnesium, inorganic phosphorus, total protein, lactate dehydrogenase, uric acid.

The liver function tests: ACT/cast, ALT/SALT, alkaline phosphatase, GGT, albumin, bilirubin (direct, total)

Lipid profile: total cholesterol, triglyceride, HDL, LDL

The study of coagulation international normalized ratio (OLS) prothrombin time (PT), activated partial prothrombin time (aPTT)

Urinalysis: glucose, ketones, blood, pH, protein, nitrite and leucocytes (can be made microscopic examination, if there are deviations from the norm for blood, nitrite and/or leukocytes)

The study of viral hepatitis: serology surface antigen of hepatitis b (HBsAg) and hepatitis C virus (HCV) (at screening or clinical detection during the study)

Research autoimmune hepatitis: antibody to smooth muscle (ATGM), antinuclear antibodies (AAA), antibody to LCMI, antibody to soluble liver antigen (RPA)

Antibody to the drug: antibody to SBC-102

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

The pregnancy test: All women d�should at least once a month to undergo pregnancy testing. The tests are performed using serum during visit 1 and visit 8 and urine during visit 2 and visit 6.

Farmacocinetica assessment (FC): FC samples are taken from the shoulder opposite to the catheter for infusion. Performed intensive sampling for measurement of serum level of SBC-102 at day 0 (dose 1, visit 2) and day 21 (dose 4, visit 6): just before the introduction of dose (within 30 minutes of the dose); 10(±1), 15(±1), 20(±1), 40(±2), 60(±2) and 90(±2) minutes during the infusion and at the end of the infusion (approximately 120 minutes); and through 5(±1), 10(±1), 20(±1), 30(±1), 40(±2), 60(±2) and 120(±2) after completion of the infusion.

Preparation of SBC-102

The weight of the subject recorded during visit 1, is used to calculate the volume of SBC-102 for each infusion. Medicinal product SBC-102 for intravenous infusion is prepared by dilution using the following steps:

1. The tubes are extracted from the refrigerator.

2. It is confirmed that the expiration date printed on the tube, not expired.

3. Is determined by the calculated total SBC-102 is required to administer the dose Example:

Weight subjects (in kg): 70 kg

The dose level for the subject: 3 mg·kg-1

The concentration of the drug: 2.0 mg·ml-1

1. Calculation of dose for the subject is:

2. Calculation of dose injection:

4. Follow�their infusion packages with 0.9% saline used based group dosing:

GroupDose (mg·kg-1)The volume of infusion package (ml)
10,35100
21
33250

5. Volume that is equivalent to SBC-102, desired for administration (in accordance with the calculations in step 3 above) is extracted from 100 ml or 250 ml infusion package with 0.9% saline (i.e., using the example above, 105 ml brine is extracted from a 250 ml infusion bag).

6. The calculated total volume of SBC-102 is retrieved and transmitted to the infusion package with 0.9% saline (i.e., using the example above, 105 ml of a solution of SBC-102 is extracted and transferred to an infusion bag).

7. For mixing gently turned over the package.

The introduction of recombinant CFL

1. Tube for intravenous infusion is connected to the package diluted with SBC-102.

2. The tube is filled and all the air comes out.

3. The flow regulator of infusion set to the introduction of the total volume at the following speeds for about 100 minutes:

GroupDose (mg·kg-1)The rate of infusion(hour)The rate of infusion (min)
10,3560 ml1 ml
21
33150 ml2.5 ml

4. Select a section intravenous infusion, which varies by subject and may include the veins in front of the elbow joint or wrist vein (or Central venous catheter).

5. Tube for intravenous infusion is connected to angiocatheter. Saline solution is injected into the IV line to access the vessels and check easy passage of saline solution.

6. Drip tape is fixed.

7. The infusion of SBC-102 starts, it uses the speed control flow.

8. The infusion is regularly monitored.

9. When the package is emptied, 25 ml of 0.9% saline solution immediately injected into the infusion bag, using port injection.

10. The dropper installs the same rate of infusion (60 ml per hour [1 / minute] for the dose of 0.35 mg·kg-1and mg·kg -1and 150 ml / hour [2.5 ml per minute] for the dose of 3 mg·kg-1) until the completion of the infusion. The end of the infusion is determined when the infusion and rinsing is completed and documented.

Infusion reactions

Reactions related to the infusion (ROS), defined as any unwanted immune phenomenon that, at least possibly related to the infusion. The DITCH is classified as acute (manifesting within 24 hours after the start of infusion) or delayed (reflected in 1-14 day after injection).

Medicines and equipment for the treatment of hypersensitivity reactions should be ready for immediate use in case of unexpected adverse hypersensitivity reactions. These tools include, but are not limited to, oxygen, acetaminophen, antihistamines (eg, diphenhydramine, parenteral and oral drugs), corticosteroids, epinephrine (adrenaline) and apparatuses for restoration of cardiac activity and respiration.

Signs of possible acute DITCH may be classified as follows: hyperemia, congestion, fever and/or chills, nausea, itching, urticaria, gastrointestinal symptoms (vomiting, diarrhea, abdominal cramps), cardiopulmonary reactions, including chest pain, shortness of breath, wheezing, stridor, hypotension or hypertension. If any of the above signs or symptom�to be observed during the infusion and the subject remains hemodynamically stable, the rate of infusion should be decreased or the infusion should be stopped. If the subject continues to show signs of hypersensitivity, dose of antihistamine may be administered intramuscularly or slowly intravenously. Subjects with serious infusion reactions with clinically significant cardiovascular or respiratory effects the infusion is terminated. Under such anaphylactic reactions of a subject to be treated by antihistamines, corticosteroids and epinephrine.

Example 8

The introduction of recombinant CFLs in the body of the rat

The impact of repeated doses of recombinant CFLs person on weight, triglycerides and cholesterol in tissue, hepatomegaly, splenomegaly, lymphadenopathy, abdominal mass, and other parameters were evaluated in rats Donryu deficit CFLs described in Yoshida and Kuriyama (1990) Laboratory Animal Science, volume 40, pp. 486-489 (see also Kuriyama and others (1990) Journal of Lipid Research, vol. 31, pp. 1605-1611; Nakagawa et al., (1995) Journal of Lipid Research, vol 36, pp. 2212-2218), the disclosure of which is included in this application in its entirety by reference. At 4 weeks of age, rats of Donryu, which is homozygous for deletions CFL (CFL-/-), were assigned into groups for the introduction of recombinant CFLs person produced in the oviduct of transgenic chicken, or for doses of saline placebo. Rats of the same litter wild type were used in �the quality control. Rats with CFL-/-administered dose once a week for four weeks (a total of four doses) or once every two weeks for four weeks (a total of two doses) by injection in the tail vein as a single dose or in two equal doses with an interval of 30 minutes. Dose recombinant CFL was 1 mg/kg or 5 mg/kg. Schedule of doses shown in Table 5. Rats were prepared with the use of diphenhydramine (5 mg/kg) to prevent possible anaphylactic reactions and procedures based on previous experience fermentopatiami therapy in animals for the treatment of lysosomal storage diseases (Shull and others (1994) Proceedings of the National Academy of Science, Volume 91, c.12937; ilik and others (1999) The Journal of Biological Chemistry, 274, c.36335; Vogler and others (1999) Pediatric Research, 45, c.838), the disclosure of which is included in this application in its entirety by reference.

Figure 9 illustrates the daily progress in the weight gain of rats injected with the dose of 1 mg/kg recombinant CFL once a week or 5 mg/kg recombinant CFLs once in two weeks. The figure shows little difference or lack thereof in therapeutic effect between size and frequency of the two doses.

Table 5
Schedule of weighing and injection of doses to rats with deficiency CFL
Age daysPerformed evaluation / injection
Weighted
Day 13
Day 14
Day 20
Day 21Cessation of milk feeding
Day 24
Day 25
Day 27
Day 28The first injection for administration once a week and once in two weeks
Day 31
Day 32
Day 34
Day 35The second injection for administration once a week
Day 38-
Day 39
Day 41
Day 42The third injection is for administration once a week; a second dose for administration once every two weeks
Day 45
Day 48
Day 49Fourth dose for administration once a week
Day 55
Day 56Autopsy

Example 9

Pathological study of rats with CFL-/-with recombinant CFL

After the termination of the study described in Example 8, the animals were sacrificed and subjected to autopsy for the study of General pathology, histopathology and clinical biochemistry.

Common autopsy included a study of the outer surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. The mass of internal organs and tissues was determined for rats, and organs and tissues were collected and placed in 10% formalin neutral buffer. After fixation, tissues were processed for histological preparations stained with hematoxylin and stained sections were prepared and evaluated.

Example 10

Internalization of recombinant CFLs person in the lysosomes of macrophage and fibroblast

The ability of recombinant CFLs person produced in transgenic bird ("SBC-102"), link cells and be internaitional in the lysosomal compartment was investigated in vitro using cells of macrophages and fibroblasts. When cultured with cells macrophages revealed that SBC-102 with a fluorescent tag was located next to the lysosomes. This effect can be reduced by using a competitor polysaccharide mannose, considering the receptor N-acetylglucosamine/mannose as a mechanism for the recognition and uptake by these cells. SBC-102 has increased activity in CLL associated with cells, in fibroblasts che�of owaka deficit CFL and normal fibroblasts of rats after in vitro cultivation, by showing that the impact of SBC-102 can lead to significant replacement of deficient enzymatic activity.

Mannose-6-phosphate (MR) is present in the structures of the oligosaccharide chains SBC-102, which have been involved in the delivery of lysosomal enzymes to the cells through different types of ubiquitous receptor MP6.

Recombinant CFL was purified from egg white of the transgenic chicken. Oregon Green NHS was obtained from Invitrogen™ (#0-10241). Line the alveolar macrophage, NR8383, line and mouse fibroblast, NIH-3T3, were received from ATS. The Wolman fibroblasts deficit CFLs were obtained from the Institute of medical research, Coriell, and LysoTracker® Red was obtained from Invitrogen™.

Labelling of enzymes: 4 mg of CFLs produced in transgenic bird, FSB were labeled with Oregon Green, in accordance with the manufacturer's recommendations, and the reaction was then detalizirovano in PBS, and then concentrated.

The macrophage uptake: CFL with a fluorescent tag, produced in transgenic bird (5 µg/ml), and LysoTracker® Red was cultivated with NR8383 cells within 2 hours. The cells were investigated using defocuses fluorescence microscopy using a mode of sequential scanning at a wavelength of 488 nm, and then 514 nm.

Competitive inhibition by mannan: SBC-102 with a fluorescent tag (5 μg/ml) and mannan cultivation�again with NR8383 cells within 2 hours. Cells were trypsinization and absorption of recombinant CFL was measured by sorting fluorescently activated cell using the median intensity of fluorescence as an endpoint.

The ability of CFLs produced in transgenic bird, be absorbed and then be incorporated into lysosomes of target cells was investigated using cell lines of macrophage, NR8383. CFLs with a fluorescent tag, produced in transgenic bird and the lysosomal marker, "LysoTracker® Red (Invitrogen™) were cultured with the cells for 2 hours. The close location of CFLs produced in transgenic bird and lysosomal marker in the lysosomes of these cells was then investigated by defocuses microscopy, using a sequential scan mode (Figure 11). Recombinant CLL demonstrated a close proximity to the lysosomes, which is consistent with a similar pattern in the study in vitro using recombinant CFLs person from different sources.

Communication ability of CFLs produced in transgenic bird, with the receptor N-acetylglucosamine/mannose was assessed through analyses of competitive communications using cellular macrophage line, NR8383 (Figure 12). CFLs with a fluorescent tag (or Green) produced in transgenic bird (5 µg/ml), and �various concentrations of mannan oligosaccharide containing mannose were cultured with the cells for 2 hours. The relative inhibition of the uptake of CFLs produced in transgenic bird, mannan when compared with the product without mannan was quantified by sorting fluorescently activated cell using the median intensity of fluorescence as an endpoint. Observed inhibition, dose-dependent mannose, contact/absorption, CFLs produced in transgenic bird, which is consistent with the interaction of CFLs produced in transgenic bird, with N-acetylglucosamine.

In addition, the uptake of mannose-6-phosphate in the fibroblast cells was demonstrated in competition experiments with mannose-6-phosphate.

Example 11

Increased activity of CFLs in cells exposed to treatment CLA, catalyzes the hydrolysis of cholesterol esters and triglycerides to free cholesterol, glycerol and free fatty acids. Thus, the activity of CFLs can be measured, for example, by splitting fluorogenic substrate oleate 4-methylumbelliferyl (MWA).

Fibroblasts

The ability of the impact of CFLs produced in transgenic bird, the increased activity of CFLs was investigated using normal cells and cells with deficiency of CFLs in artificial conditions. Fibroblasts were isolated from a patient with Wolman disease and normal rat fibroblasts (NIH-3T3) cultevero�of ALIS in the presence of CFLs, produced in transgenic bird, at concentrations of 0, 0.16 points or 0.5 μg/ml for 5 hours. Then cells were washed to remove spurious signals and cell lysates were analyzed to determine the activity of CFLs, using the substrate 4 oleate-methylumbelliferyl (MWA). Figure 13 shows that the activity of endogenous CFL, associated with the cells, was lower in Wolman fibroblasts compared to NIH-3T3, and dose-dependent increase in the activity of CFLs was observed in cells of both types after cultivation with CFLs produced in transgenic birds (Figure 13).

Leukocytes

Serum mononuclear leukocytes were obtained from patients with a deficiency of CFLs before and after injection. The blood samples were kept chilled without losing enzyme activity. Mononuclear leukocytes (lymphocytes) were isolated from blood using the drug of ficoll and sodium diacetate. 4-8 ml of blood, pre-diluted 1:1 with balanced salt solution Hanks, was carefully placed onto 3 ml of ficoll-Pak and centrifuged. The ring of mononuclear cells was aspirated and washed once with Hanks solution, and then at least twice resuspending sediment in 1-2 ml of water. The pellets were frozen at -20°C before use. Before analysis the pellets were thawed, resuspendable in distillirovanna�th water and podvegnut the influence of ultrasound on the ice. The preparation was then centrifuged at 20000 × g for 15 minutes at 4°C. the Supernatant (containing 0.5-1.5 mg protein/ml) were stored on ice until analyzed.

The substrate is acidic lipase was prepared by adding 1 ml of 10 mm MOO (4 oleate-methylumbelliferyl) in hexane to 1 ml of 16 mm L-α-phosphatidylcholine in CHCl3. The solvents were evaporated in the presence of N2and 25 ml of 2.4 mm taurodeoxycholic acid (sodium salt) in water was added. The mixture was subjected to ultrasound on ice for 1-2 minutes at 30-40 watts. Before analysis 1 volume of substrate was diluted with 7 volumes of 200 mm buffer sodium acetate /acetic acid (pH of 4.0). Each 2 ml of the reaction cuvette contained 100 nmol oleate 4-methylumbelliferyl, 160 nmol L-α-phosphatidylcholine and 600 nmol taurodeoxycholate sodium.

The reaction was initiated by addition of 5-100 ál of enzyme and monitored at 37°C, using spectrofluorometer. It was discovered spina MOO, for example, excitation at a wavelength of approximately 360 nm and emission at about 460 nm released fluorophore, 4-methylumbelliferone (MU). The change in fluorescence with time was recorded.

Example 12

Analysis of recombinant CFL (SBC-102) under natural conditions

Rat Joshila (i.e. homozygous) (see Kuriyama and others (1990), Journal of Lipid Research, vol. 31, pp. 1605-1611; Nakagawa et al., (1995) Journal of LipidResearch, vol. 36, pp. 2212-2218; and Yoshida and Kuriyama (1990) Laboratory Animal Science, volume 40, pp. 486-489) deficit CFLs were tucked treatment through the introduction of SBC-102 (5 mg/kg intravenously) or placebo once weekly for 4 weeks starting at 4 weeks of age. Each introduction SBC-102 has been injected in the tail vein of the mouse in two equal doses (2.5 mg/kg) every 30 minutes. Rats and control individuals of the same age were studied one week after the last dose. The tests were performed three times.

Common pathological examination of animals after treatment with SBC-102 showed normalization of liver color in addition to reducing the size of the body. Rats after treatment with SBC-102 showed essentially normal histology of the liver in marked contrast with the considerable accumulation of foamy macrophages in animals injected with placebo. Serum levels of alanine and aspartic acid transferase, elevated in rats with CFL-/-were also reduced in rats after treatment with SBC-102.

The mass of internal organs and tissues was determined for each rat, and the data shown in Figure 14. The size of the authority are presented as percentage of body weight, determined at 8 weeks of age rats with CFL-/-and rats with CFL+/+after weekly, placebo or SBC-102 5 mg/kg for 4 weeks.

Weight bodies� rats Yoshida after treatment with SBC-102 or placebo was compared with the weight of wild-type rats, as shown in Figure 15. SBC-102 (5 mg/kg) or placebo was administered intravenously in a single dose or in divided doses (within a 4 hour period of time) to rats with CFL-/-. Rats with CFL+/+was a control specimen of the same litter.

Example 13

Analysis of triglycerides

Analysis of triglycerides was performed on liver tissue and spleen taken from homozygous wild-type animals after administration of placebo or SBC-102. Analyses of triglycerides were carried out using standard methodologies (i.e. International directory of sets of quantitative determination of triglycerides MBL JM-K622-100) and were performed three times.

Table 6
Triglyceride levels in the liver and spleen in rats with wild-type deficiency CFL
Triglyceride (mg/mg wet tissue)
Wild type (n=3)Placebo (n=3)SBC-102 (n=3)
Liver488457
Spleen322 4

The level of the substrate of the liver

Figure 16 shows the levels of cholesterol, cholesterol ester and triglyceride in the liver, certain rats at 8 weeks of age with BV and a deficit after the weekly CFL, placebo or SBC-102 5 mg·kg-1for the past 4 weeks.

Example 14

To study the response of rats at the dose was given

Based on the studies described above, the pharmacodynamic effects (PD) of a range of doses and schedules of administration (once a week and once in two weeks) doses of CFLs ("SBC-102") were investigated in rats with CFL-/-. In these studies of SBC-102 was administered as an intravenous injection at doses of 0.2, 1, 3 and 5 mg/kg, once every two weeks, or 0.35, 1.0 and 5.0 mg/kg, once a week for one month, starting at 4 weeks of age. The results show improvement in increasing the body weight (BW) (Figure 17) organomegaly (Figure 18) and in the levels of substrate (Figure 19). The level of serum transaminase levels also decreased with increasing dose of SBC-102 and reached essentially level for the wild type at higher doses.

Example 15

The pharmacokinetics of SBC-102

and. Sampling

FC samples were collected from adult patients suffering from a deficit of CFLs with late onset. Patients received doses of 0.35 mg/kg for 2 hours. Serum samples on day 0 (dose 1, visit 2) and day 21 (dose 4, visit 6) were from�wounds just before the introduction of dose (within 30 minutes of the dose); through 10(±1), 15(=±1), 20(=±1), 40(±2), 60(±2) and 90(±2) minutes during the infusion (BB) and at the end of the infusion (KB) approximately 120 minutes after the start of infusion); and through 5(±1), 10(±1), 20(±1), 30(±1), 40(±2), 60(±2) and 120(±2) minutes after completion of infusion (PV).

b. Analysis of serum enzyme

4-Muo (4 mm) and stored in a freezer at -20°C, was thawed at 4°C in the refrigerator in the dark and placed in an incubator at 25°C for 1.5 hours in the dark before use. The standard was prepared by dilution of the medicinal product SBC-102 to 1.56 ng/ml of Neutral buffer for analysis was added. All samples were diluted to 50 ng/ml for the first dilution. Standards and samples were sown in the tablet immediately after dilution. After preparation of standards and samples at 62.5 ál of buffer for analysis (0,2 mol/l of trihydrate sodium acetate, pH 5.5) was added to each well. 12,5 ál of standards and samples were added to each well with redundancy. 4 - Muo (4 mm) was diluted to 1.6×4% solution of Triton X-100 and added to 25 μl in each well. Mnogolinkovuyu tablet, hermetically closed, was shaken several times to mix and then was placed in an incubator at 37°C for 30 minutes. After incubation 50 ál of stop solution (0,77 M Tris pH 8.0) was added to each well to obtain a final volume of 150 µl/well. The tablet was placed on the card�the crucial device for microplates, and the levels of fluorescence were measured from the bottom of the tablet at excitation 360 nm and emission 460 nm.

As shown in Tables 7-11, serum Cmax of recombinant CFL, administered to adult patients suffering from a deficit of CFLs with late onset, were in the range of from about 270 ng/ml to 720 ng/ml half-life (t1/2) ranged from 7.6 minutes to 16.7 minutes, the average value of the half period t1/2was about 13 minutes (standard deviation equal to 3,812).

Table 7
ID of the patient
(Dose: 0.35 mg/kg)
Visit No.Concentration (ng/ml)Nominal time
02-00125,63Before infusion
02-0012241,8810 min BB
02-0012369,4415 min BB
02-0012369,9320 min BB
02-0012359,6440 min BB
02-0012294,6460 min BB
02-001271,8590 min BB
02-001271,20KB
02-001237,955 min MF
02-001224,9110 min PV
02-001214,1620 min MF
02-00129,7630 min MF
02-00129,0240 min MF
02-00127,2060 min MF
02-00127,51 120 min MF
Cmax=369,93 ng/ml; t1/2=16,8 min; NPCA=4,68 ng/ml

Table 8
ID of the patient
(Dose: 0.35 mg/kg)
Visit No.Concentration (ng/ml)Nominal time
03-00128,23Before infusion
03-0012199,8010 min BB
03-0012215,1015 min BB
03-0012228,1220 min BB
03-0012237,2540 min BB
03-0012210,7360 min BB
03-0012262,4190 min BB
03-0012102,39KB
03-001252,205 min MF
03-001233,7510 min PV
03-001217,6020 min MF
03-001212,1730 min MF
03-001210,8240 min MF
03-00129,3960 min MF
03-00128,72120 min MF
Cmax- 262 ng/ml; t1/2=15,3 min; NPCA=4,68 ng/ml

Table 9
ID of the patient
(Dose: 0.35 mg/kg)
Visit No.Concentration (ng/ml) Nominal time
03-0022<4,68Before infusion
03-0022480,5510 min BB
03-0022531,1615 min BB
03-0022613,8520 min BB
03-0022717,7540 min BB
03-002284,4660 min BB
03-002254,3490 min BB
03-0022171,41KB
03-002287,955 min MF
03-002249,7010 min PV
03-002 216,4720 min MF
03-002211,0130 min MF
03-00229,7640 min MF
03-00226,2860 min MF
03-00225,19120 min MF
Cmax=718 ng/ml; t1/2=10,8 min; NPCA=4,68 ng/ml

Table 10
ID of the patient
(Dose: 0.35 mg/kg)
Visit No.Concentration (ng/ml)Nominal time
02-00165,12Before infusion
02-0016249,9110 min BB
02-0016 294,3715 min BB
02-0016313,7520 min BB
02-0016330,0440 min BB
02-0016245,4860 min BB
02-0016262,7890 min BB
02-001681,09KB
.02-001632,105 min MF
02-001620,3910 min PV
02-001610,7420 min MF
02-00168,0830 min MF
02-00166,3840 min MF
02-00165,8260 min MF
02-0016<4,68120 min MF
Cmax=330 ng/ml; t1/2=15,3 min; NPCA=4,68 ng/ml

Table 11
ID of the patient
(Dose: 0.35 mg/kg)
Visit No.Concentration (ng/ml)Nominal time
03-0016Of 6.63Before infusion
03-0016317,4510 min BB
03-0016333,4615 min BB
03-0016310,8320 min BB
03-0016378,6840 min BB
03-0016 234,4960 min BB
03-0016246,0790 min BB
03-0016206,06KB
03-0016253,805 min MF
03-001670,8110 min PV
03-001624,6120 min MF
03-001612,3230 min MF
03-00169,0340 min MF
03-00167,1860 min MF
03-0016The 5.56120 min MF
Cmax- 379 ng/ml; t1/2=7,7 min; NPCA=4,68 ng/ml

Example 16

Analysis of immunogenicity: Measurement�of antibodies to SBC-102

Unknown sample, a positive sample and a negative sample were diluted 1:20 in 5% dry milk in 1 × PBS and were treated at 4°C for 12-18 hours on a rotator (500 rpm). Before analysis, the samples were centrifuged at 2000 × g for 20 minutes and the supernatant was transferred into a new 1.5 ml tube.

SBC-102 has been diluted in IX PBS to a concentration of 0.5 μg/ml, and 100 μl was placed in each well of the tablet ELISA with 96 wells. The tablet was closed adhesive cover and were treated at room temperature for 8 hours or overnight at 4°C. After incubation the wells were washed three times with 1x buffer for washing. 200 μl 5% BSA-IgG was added to each well and the plate was sealed and treated at 4°C for 12-18 hours or at room temperature for 2 hours. After incubation the wells were washed three times with 1x buffer for washing. 100 µl of a control sample, the unknown sample and a negative sample were added into the wells three times. Tablet were treated at room temperature for 1.5 hours on the device for shaking microplates (500 rpm). After incubation the wells were washed three times with 1x buffer for washing. 100 µl biotinylating SBC-102, diluted in the buffer for dilution to a concentration of 100 ng/ml, was added to each well, and the tablet in�was oberaula at room temperature for 1.5 hours on the device for shaking microplates (500 rpm). After incubation the wells were washed three times with 1x buffer for washing. 100 μl of the composition of streptavidin-renasterea peroxidase, diluted 1:4000 in the buffer for dilution, was added to each well. Tablet were treated at room temperature for 1.5 hours on the device for shaking microplates (500 rpm). After incubation the wells were washed four times in 1x buffer for washing. 100 μl of TMB substrate was added to each well, and the tablet were treated for 15 minutes in the dark. 50 µl of stop solution (0.5 N H2SO4) was added to each well to stop the reaction. OD was measured at a wavelength of 450 nm.

As shown in Table 12, patients treated with weekly doses of 0.35 mg/kg of SBC-102 for 4 weeks did not exhibit elevated levels of antibodies to SBC-102, and it can be assumed that enzyme replacement therapy by infusion of SBC-102 does not cause any significant immunogenicity in human patients. These patients do not demonstrate any adverse events or reactions related to infusion (ROS).

Table 12
SampleThe mean value ODStandard deviation. SBC-102 Concentration (ng/ml)
Neg. Counter.0.056N/00
Position. Counter. 10.0760.00515.6
Position. Counter. 20.0900.00331.2
Position. Counter. 30.1320.00862.5
Position. Counter. 40.2010.009125
Position. Counter. 50.3490.019250
Position. Counter. 60.6350.012500
Position. Counter. 70.9580.1011000
ID of the patient The mean value ODStandard deviation
01-001 Visit 10.0530.000
02-001 Visit 10.0510.000
02-001 Visit?0.0530.002
02-001 Visit 80.0500.001
03-001 Visit 10,0510.001
03-001 Visit 20.0510.001
03-001 Visit 70.0500.000
03-002 Visit 10.0520.004
03-002 Visit 20.0560.006

Example 17

Treatment of Wolman disease (WD) the introduction of recombinant CFL

At 7 weeks of age the patient-a girl sent to the hospital for breach of weight gain, and weak progress from the day of birth. Upon initial physical examination, the patient weighs 3.6 kg(birth weight of 3.7 kg) and thin wrinkled skin. The abdomen was distended, with a strong 6 cm hepatomegaly and marked splenomegaly approximately 4 cm Enlarged lymph nodes noted in the groin area, muscle activity is weak.

The initial level of hemoglobin is 9.2 GM, plaques - 506000 and leukocyte - 11550. Urine analysis normal and bone marrow smears show vakuolizirovannye lymphocytes and a large number of foam cells. Measurement of biochemical serum parameters: total lipids: 834 mg/100 ml, phospholipid: 176 mg/ 100 ml triglycerides: 141 mg/ 100 ml cholesterol: 129 mg/ 100 ml, bilirubin: 0.3 mg/ 100 ml, alkaline phosphatase: 9.0 FUCKED %, SGOT: 90 units, SGPT: 50 units, cholinesterase: 20 units, nitrogen blood urea: 8.3 mg, blood sugar fasting: 45 mg/100 ml. CT scan of the abdomen shows hepatosplenomegaly and bilateral symmetric increase of the adrenal gland with calcification.

The patient is surgically implanted port access to the vascular system for administration of doses. After connecting the port to the ambulatory infusion device to the patient is entered preparatory dose diphenhydramine 1 mg/kg 20 minutes before infusion of recombinant CFLs to prevent possible anaphylactic reactions during infusion. Then the patient is injected recombinant CFLs in the form of a dose of 1 mg/kg for 5 hours by means of intravenous infusion. It �Directors repeats once every 7 days without determining the number of introductions.

Within two weeks of the first dose of recombinant CFLs is estimated to increase in weight and size of the main abdominal organs using ultrasound examination. Also performed laboratory studies of the activity of acid lysosomal lipase.

Example 18

The treatment of the disease of accumulation of cholesterol esters (BIH) the introduction of recombinant CFL

3-year-old boy with itchy abdominal irritation was examined by a pediatrician. Upon examination of the abdomen the doctor marked hepatomegaly, which is confirmed by ultrasound examination. At this point in time, no diagnosis, and the patient is observed periodically.

At the age of 8 he was sent to hospital with gastroenteritis. Light microscopy of a liver biopsy shows increased intracytoplasmic glycogen and small lipid droplets in hepatocytes. Electron microscopy shows membrane lipid drops connections with a small e-dense granules. Delivered a working diagnosis of the disease is the accumulation of type III glycogen (glycogenes type III), but the activity of skin fibroblasts from normal.

At the age of 10 hepatomegaly is present, and takes a second liver biopsy, light microscopy shows a modified lobular architecture of the liver parenchyma with loosened by hepatocytes, containing�their cytoplasmic granules and vacuoles with mild periportal fibrosis. Discovered that the activity of acid lipase fibroblast is 7% of normal activity, which confirms the diagnosis BIH. Plasma concentrations of total cholesterol (OH), triglycerides (TG), cholesterol of low density lipoproteins (LDL's) above the 95th percentile for age and sex when 7,51, 3,24 and 5.58 mmol/l, respectively, while the concentration of cholesterol of high-density lipoprotein (PVP's) in the plasma below the 5th percentile when 0,47 mmol/l; it combines hyperlipidemia (hypercholesterolemia, hypertriglyceridemia, hypoalphalipoproteinemia and hyperbetalipoproteinemia).

The patient is surgically implanted port access to the vascular system for administration of doses. After connecting the port to the ambulatory infusion device to the patient is entered preparatory dose diphenhydramine 5 mg/kg 20 minutes before infusion of recombinant CFLs to prevent possible anaphylactic reactions during infusion. Then the patient is injected recombinant CFLs in the form of a dose of 5 mg/kg for 5 hours by means of intravenous infusion. This treatment is repeated once every 14 days without specifying the number of injections.

Within two weeks of the first dose of recombinant CFLs is estimated to increase in weight and size of the main abdominal organs using ultrasound examination. Also performed laboratory�rye research activity of acid lysosomal lipase.

Each example in the above description is provided to explain the invention and not a limitation of the invention. Indeed, a specialist in this field will be obvious that various modifications, combinations, additions, deletions, and variations can be made in this invention without departing from the scope or the spirit of the invention. For example, the properties illustrated or described as part of one variant of the embodiment can be used in another embodiment to obtain a further variant embodiment. It is implied that the present invention covers such modifications, combinations, additions, deletions and variations.

All publications, patents, patent applications, Internet sites and access numbers/sequences in the database (including both polynucleotide and polypeptide sequences) cited in this application are included in this application in its entirety by reference for all purposes to the same extent as if each individual publication, patent, patent application, Internet site or a number/sequence in the database were specifically and individually indicated to incorporation by reference.

1. A method of treating a patient-a person with deficiency of acid lysosomal lipase (CFLs), which includes introduction to the specified patient is a human recombinant CFL che�of owaka in number, effective to reduce the level of liver transaminases in serum or blood to a normal level, the above-mentioned recombinant CFLs person is administered once about every 7 days to once every 30 days and specified introduction is sufficient to reduce liver injury in the specified patient person.

2. A method according to claim 1, characterized in that the specified transminase liver selected from the group consisting of the serum aspartate aminotransferase and alanine aminotransferase (ALT).

3. A method according to claim 1, characterized in that the specified transminase liver is an ACT.

4. A method according to claim 1, characterized in that the specified transminase liver is an ALT.

5. A method according to claim 1, characterized in that said introduction is sufficient to improve hepatomegaly.

6. A method according to claim 1, characterized in that said introduction is sufficient to increase the level of serum hemoglobin.

7. A method according to claim 1, characterized in that said administration is sufficient to reduce the size of the liver.

8. A method according to claim 1, characterized in that said administration is sufficient to reduce the level of serum ferritin.

9. A method according to claim 1, characterized in that the specified recombinant CFLs person is administered once about every 7 days.

10. A method according to claim 1, characterized in that the specified recombinant CFLs person is administered once about every 14 days.

11. A method according to claim 1, wherein the human patient suffers from Wolman disease.

12. A method according to claim 1, wherein the human patient suffers a disease of accumulation of cholesterol esters.

13. A method according to claim 1, characterized in that said recombinant CFLs person includes at least one terminal mannose or at least one terminal mannose-6-phosphate.

14. A method according to claim 1, characterized in that the specified amount, effective to reduce the level of liver transaminases in serum or blood to a normal level, is approximately 1 mg per kilogram body weight of the specified patient person.

15. A method according to claim 1, characterized in that the half-life (t1/2) the indicated recombinant CFLs human serum is less than about 20 minutes.

16. A method according to claim 1, characterized in that the half-life (t1/2) the indicated recombinant CFLs human serum is approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 minutes.

17. A method according to claim 1, characterized in that the Cmaxthe indicated recombinant CFL humans is from about 200 ng to about 800 ng per ml of serum.

18. A method according to claim 1, characterized in that the Cmaxspecified a recombinant human�a combined CFLs person is at least 200 ng per ml of serum.

19. A method according to claim 1, characterized in that the specified recombinant CFLs person injected.

20. A method according to claim 19, characterized in that the specified recombinant CFLs person is administered by infusion.

21. A method according to claim 20, characterized in that said infusion is indicated for the patient-the person performing for the time from about one to about four hours.

22. A method according to claim 1, characterized in that said administration is sufficient to reduce lymphadenopathy.

23. A method according to claim 1, characterized in that the specified age of the patient-the person less than one year and the specified introduction is enough to increase the growth rate of a specified patient person.

24. A method according to claim 1, further comprising introducing a second drug.

25. A method according to claim 24, characterized in that said second drug is a means of lowering cholesterol.

26. A method according to claim 25, characterized in that said second drug is a statin.

27. A method according to claim 25, characterized in that said second drug is an ezetimibe.

28. A method according to claim 24, characterized in that said second drug is an immunosuppressant.

29. A method according to claim 24, characterized in that said second Leka�only remedy is an antihistamine.

30. A method according to claim 29, characterized in that the antihistamine is diphenhydramine.

31. A method according to claim 30, characterized in that the specified diphenhydramine is administered in an amount of from about 1 to about 5 mg per kilogram of body weight of the specified patient person.

32. A method according to claim 30, characterized in that the specified diphenhydramine administered for from about 20 to about 90 minutes prior to the introduction of recombinant CFLs person.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to prodrug preparations of glucagon superfamily peptides, in which the glucagon superfamily peptide is modified by binding a dipeptide with the peptide of the glucagon superfamily by an amide bond.

EFFECT: produgs, disclosed in the claimed invention, have an increased half-life and transfer into an active form in physiological conditions as a result of a non-enzymatic reaction, caused by chemical instability.

30 cl, 15 dwg, 8 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: invention provides a solid hypolipidemic dosage form containing rosuvastatin or its pharmaceutically acceptable salt in an amount of 3 to 15%, processing additives and a pharmaceutically acceptable excipient containing microcrystalline cellulose, lactose monohydrate, polyvinylpyrrolidone and croscarmellose sodium. The above excipient represents granulate in an amount of 79 to 95 wt % of the dosage form containing absorbed moisture within the range of 0.5% to 1.5%. What is also described is a method for preparing the dosage form.

EFFECT: uniform distribution of the active substance and storage stability of the dosage form of rosuvastatin.

11 cl, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to a compound of formula (I) or its pharmaceutically acceptable salts of formula (I), wherein X represents O, S; Y represents O, S; R1 independently represents H, alkyl; G1 represents ethyl; each G2 and G3 are independently specified in H, alkyl, trifluoromethyl, halogen, nitro, amido, cyano and tetrazolyl. The invention also refers to a pharmaceutical composition possessing activating action on peroxisome proliferator activated receptors subtype α, subtype δ and subtype γ and containing an effective amount of the compound of formula (I) or its pharmaceutically acceptable salts. The compounds of formula (I) are applicable for treating or producing a drug preparation for treating or preventing the diseases associated with peroxisome proliferator activated receptors subtype α, subtype δ and subtype γ. The compounds of formula (I) are produced by a reaction of the compound of formula (III) and the compound of formula (IV) when heated in acetonitrile under reflux in the presence of potassium carbonate to produce the compound of formula (II), to saponify the compound of formula (II) in alcoholic solution in the presence of alkali and to acidify the reaction mixture to produce the compound of formula (I). X, Y, R1, G1, G2 and G3 have the above values; R3 represents a leaving group specified in OH, Cl, Br, I, OTs, OMs.

EFFECT: compounds of phenylpropionic acid possessing the activating action on peroxisome proliferator activated receptors (PPARα, δ, γ).

15 cl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I, wherein R1 and R2 are identical or different and specified in an alkyl or alkenyl hydrocarbon chain; the R3 group values split by lipase are specified in the patient claim. R4 and R5 are independently hydrogen or C1-C7alkyl; R6 represents hydrogen or C1-C7alkyl; and R7 and R8 are independently hydrogen or C1-C7alkyl. The invention also refers to using compounds of formulas ,

which are introduced into the mammalian biological system and increase the cell concentrations of specific sn-2 substituted ethanolamine-plasmalogens.

EFFECT: compounds are applicable in treating or preventing the age-related disorders associated with high membrane cholesterol, high amyloids and low plasmalogens, such as neurodegeneration, cognitive disorder, dementia, cancer, osteoporosis, bipolar disorder and vascular diseases.

11 cl, 18 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of general formula I, or their racemic mixture, or their individual optic isomers, or pharmaceutically acceptable salts possessing the properties of TGR bile acid receptor agonist. The invention also refers to methods for preparing the compounds. In general formula I , X represents amino group R'R"N, wherein the substitutes R' and R" can be optionally identical, or represents hydrogen, C1-C6alkyl, C3-C6cycloalkyl; substituted C1-C6alkyl, wherein the substitute is specified in phenyl or phenoxy, each of which can be substituted by halogen in turn, C1-C3alkyl, C1-C3alkoxy, phenyloxy, C3-C6cycloalkyl, 5-6-merous heteroaryl with 1 nitrogen atom; aryl specified in phenyl optionally substituted by fluorine, C1-C3alkyl, C1-C3 alkoxy; 5-6-merous heteroaryl with nitrogen atom as heteroatom; C2-C4alkenyl, acyl specified in C1-C6alkylcarbonyl or C3-C6cycloalkylcarbonyl; or substituted oxygroup, which represents hydroxy group, wherein hydrogen is substituted by C1-C6alkyl optionally substituted by hydroxy, di(C1-C3alkyl)amino, phenyl, which can be substituted by halogen in turn, C1-C3alkyl, C1-C3alkoxy; C2-C4alkenyl; and 5-6-merous heterocyclyl with nitrogen atom, or sulphur atom, or oxygen atom as heteroatom; R1a and R1b represents hydrogen, C1-C3alkyl, or R1a and R1b together form methylene chain -(CH2)n-, wherein n=2-5; R1c and R1d represents hydrogen, C1-C3alkyl; R2 represents acyl group specified in C1-C6alkylcarbonyl, wherein alkyl can be substituted by phenyl or phenoxy, each of which can be substituted by halogen in turn, C1-C3alkyl, C1-C3alkoxy; C3-C6cycloalkylcarbonyl; phenylcarbonyl, which can be substituted by halogen, C1-C3alkyl, C1-C3alkoxygroup, oxygroup, C1-C3alkylene dioxygroup; 5-6-merous heteroarylcarbonyl with nitrogen atom, or oxygen atom, or sulphur atom as heteroatom, optionally substituted by carboxy, halogen or C1-C3alkoxycarbonyl, substituted aminocarbonyl group, wherein the substitute can be specified in C1-C6alkyl optionally substituted by C1-C3alkoxycarbonyl, halogen, 5-6-merous heteroaryl together with nitrogen atom, or oxygen atom or nitrogen atom as heteroatom; C3-C6cycloalkyl; phenyl optionally substituted by halogen, C1-C3alkyl, C1-C3alkoxy, C1-C3alkoxycarbonyl, C1-C3alkylenedioxygroup; 5-6-merous heteroarym with nitrogen atom, or oxygen atom or nitrogen atom as heteroatom optionally substituted by carboxy, C1-C3alkoxycarbonyl; aminocarbonyl group substituted by C1-C3alkyl; sulphonyl group specified in alkylsuphonyl optionally substituted by hydroxyl group, cyano group, phenyl, which is optionally substituted by C1-C3alkyl, halogen, C1-C3alkoxy group; henylsulphonyl oprtionally substituted by C1-C3alkyl, halogen, C1-C3alkoxy group, cyano group, C1-C3alkylene dioxygroup, or 5-6-merous heteroarylsulphonyl with nitrogen atom, or sulphur atom, or oxygen atom as heteroatom optionally substituted by halogen, C1-C3alkyl, C1-C3alkoxy group; R3 represents hydrogen.

EFFECT: compounds can be used for preparing the pharmaceutical composition applicable in treating or preventing metabolic diseases, such as diabetes, obesity, diabetic obesity, metabolic syndrome, hypercholesterolemia, dislipidemia.

14 cl, 17 dwg, 8 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for preparing a drug substance of polyprenylphosphates and beta-sitosterol consisting in pre-mixing polyprenylphosphates and sorbitol in a mortar, a size of which fits the total volume of the mixed substances. Between a wall and/or a bottom of the mortar and the introduced polyprenylphosphates, there is a layer of sorbitol; the blended mixture of the polyprenylphosphates in sorbitol is homogenised. Sorbitol, a dry mixture of polyprenylphosphates in sorbitol in a ratio of 1:8 and beta-sitosterol are added into a homogeniser, and pulsed homogenisation is performed for 10 minutes. The homogenisation is performed at a rotation rate of 500-700 rpm for 4 minutes, 1000-1200 rpm for 2 minutes and then 500-600 rpm for 4 minutes; the rotation rate variation requires a pause of 20 minutes. Each pause of the process is followed by the intensive agitation of a mixture cup; the prepared powder is sieved at a mesh size of 20 mcm; if a sieving weight makes less than 0.1% of the weight of the loaded ingredients; the homogenisation process is terminated.

EFFECT: preparing the drug substance with the maximum effective concentration of the active substances uniformly weight-distributed and higher bioavailability.

3 cl, 2 ex, 1 tbl, 7 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, namely to a compound of formula

,

to its isomer forms and pharmaceutically acceptable salts. The invention also refers to a pharmaceutical composition based on the compound of formula I.

EFFECT: new compound increasing the HDL cholesterol concentration is prepared.

6 cl, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel substance - 2-ethyl-6-methyl-3-hydroxypyridine dichloroacetate of formula I its stable crystalline form and method of obtaining thereof. Substance (I) possesses antiatherosclerotic, hypolipidemic, antihypoxic, nootropic, anxiolytic and adaptogenic action with low toxicity (LD50=30000 mg/kg, rats, intragastrically) and absence of hygroscopicity. Substance is obtained with interaction of equimolar quantities of 2-ethyl-6-methyl-3-hydroxypyridine and dichloroacetic acid in presence of solvent.

EFFECT: increase of stability.

9 cl, 8 dwg, 10 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to heterocyclic compound of formula or to its pharmaceutically acceptable salt, where Alk represents linear C1-6 alkylene group, branched C1-6 alkylene group or C1-6 alkylene group, which has ring structure, where part of carbon atoms, constituting ring structure can be optionally substituted with oxygen atom, in ring X, X1 represents N or CRX1, X2 represents N or CRX2, X3 represents CRX3, X4 represents N or CRX4, where RX1, RX2, RX3 and RX4 each independently represents hydrogen atom; linear or branched C1-6alkyl group; linear or branched C1-6alcoxygroup; or halogen atom, in ring Y, Y1 represents CRY1, Y2 represents N or CRY2, Y3 represents N or CRY3, Y4 represents N or CRY4, RY1, RY2, RY3 and RY4 each independently represents hydrogen atom; linear or branched C1-6alkyl group, which can be substituted with halogen atom(s); C3-7alkyl group, which has ring structure; linear or branched C1-6alkoxygroup; halogen atom or cyanogroup, in ring Z, RZ represents linear or branched C1-6alkyl group, which can be substituted with halogen atom(s), or C3-7alkyl group, which has ring structure, which can be substituted with halogen atom(s). Invention also relates to particular compounds, DGAT1 inhibitor based on formula (I) compound, application of formula (I) compound, method of prevention or treatment of diseases, mediated by DGAT1 inhibition.

EFFECT: obtained are novel compounds, possessing useful biological activity.

19 cl, 19 tbl, 149 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to using the tetrapeptide Arg-Pro-Gly-Pro as an agent for preventing and treating hypercholesteremia and thrombosis.

EFFECT: using the tetrapeptide Arg-Pro-Gly-Pro leads to increasing the effectiveness of preventing and treating hypercholesteremia and thrombosis.

10 cl, 5 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: conjugate represents nanodiamond particles with size 2-10 nm with pyrophosphorase, immobilised on them by means of linker, containing amino or amide groups. Content of pyrophosphatase constitutes 0.1-1 mg per 1 mg of nanodiamond, with specific activity of pyrophosphatase constituting to 95±5% of native pyrophosphatase activity. method of conjugate obtaining includes dissolution of nanodiamond with grafted hexamethylenediamine and/or nanodiamond aminated with ammonia in water, successive addition of water buffer solution HEPES with pH 7-8, magnesium chloride, sodium fluoride, sodium pyrophosphate, pyriphosphatase, and glutaraldehyde. After that, obtained mixture is exposed for 0.5-12 h, centrifuged, washed with water buffer solution Tris-HCl and dried.

EFFECT: obtaining conjugate of nanodiamond with pyrophosphatase, possessing increased stability.

3 cl, 1 tbl, 2 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a preparatory kit presented to a patient before administering a therapeutic botulinum toxin. The declared kit contains an additive in the form of capsules or tablets, which comprise zinc ions in the organic or inorganic form in a sufficient dose of 10mg to 400mg of elementary zinc a day, and a phytase additive in an amount of 0.8-10,000 units sufficient to be administered together with the ion zinc additive for the period of zinc ion load. The preparatory kit also comprises a package insert providing the patient with recommendations to administer the zinc ion additives and to reduce the phytate dose.

EFFECT: zinc dose provides activating the therapeutic botulinum toxin for the period of zinc ion load before initiating the therapy with the therapeutic botulinum toxin.

4 cl, 1 tbl, 3 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to a composition containing a mixture of at least one proteolytic enzyme, such as subtilisin or nattokinase, and at least one lipolytic enzyme specified in a group containing lipase Cal A or Cal B of Candida anthartica, Geotrichum candidum, Candida rugosa or a mixture of these lipases to be used preventing triglyceride synthesis by splitting 2-monoacylglyceryl in the intestine, and to a combined product of the above product of the above enzymes for the above application. The technical effect consists in preventing triglyceride formation in the body under the action of the declared combination of subtilisin and lipases with the reduced blood lipoproteins maintained 8 hours after the administration thereof.

EFFECT: inventions can be used as a therapeutic agent, a cosmetic agent, a medication, a food composition, a food supplement or a nutriceutical for preventing or treating obesity, atherosclerosis, for preventing or reducing overweight.

23 cl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry and represents a composition for the external treatment and prevention of infections caused by the type 1, 2 herpes virus and bacterial complications caused by the herpetic infection, containing lysozyme, peroxidase, povyargol as active ingredients, escin and glycyrrhizinic acid or its salts as anti-inflammatory ingredients, liposomes on the basis of high-active hydrated lecithin in a combination with cholesterol as carriers and pharmaceutically acceptable carriers and excipients, with the ingredients of the composition being taken in certain proportions, wt %.

EFFECT: invention provides extending the range of products for treating and preventing the infections caused by type 1, 2 herpes virus and bacterial complications caused by the herpetic infection.

4 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to surgery. A polyethylene tube is inserted into a mesentery. A solution containing proserine in a dose of up to 50 mg is introduced through the inserted tube. A synovial fluid 2 ml dissolved in normal saline 10 ml is additionally administered along the enteroenteroanastomosis.

EFFECT: method provides preventing peritoneal adhesions following the abdominal surgical operation by administering the preparations promoting better peristalsis and slide of the intestinal loop in the abdominal cavity.

1 tbl

FIELD: medicine.

SUBSTANCE: claimed invention relates to medicine, namely to therapy, and deals with treatment of lysosomal storage disease - Niemann-Pick A or B disease. For this purpose enzyme acid sphingometlinaise is slowly, for three hours, introduced intraventricularly.

EFFECT: method and mode of introduction provide effective treatment of said disease due to reduction of pathological level of acid sphingomyelinase substrate both in tissues of brain and in visceral organs.

9 cl, 13 dwg, 5 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is described is a biodegradable haemostatic therapeutic agent for control of bleeding, which provides co-immobilising ε-aminocapronic acid 50 mg, lysozyme 5 mg in distilled water 6.5 l for 3 hours at room temperature for dialdehyde cellulose 1 g at a degree of oxidation 12%. The material is pressed out and dried to residual moisture no more than 10% in the air in darkness. After having dried, the material is milled in a fine mill to particles having a size of 20 to 50 mcm. A rate of control of bleeding is 102 seconds. A time of total resorption is 10 days.

EFFECT: agent provides a high degree of hydrolytic destruction and a good haemostatic activity.

4 cl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and may be used for improving rheological properties of sputum and inhibiting the bacterial biofilm formation in bronchi in treating cystic fibrosis. That is ensured by using recombinant human deoxyribonuclease-1 covalently bond to homopolymeric polysaccharide containing 80 links of alpha-2,8 sialic acid as an inhalation agent. There are also presented a method and administration of a drug preparation for improving the rheological properties of sputum.

EFFECT: group of inventions enables maintaining a higher level of DNA-hydrolytic activity of the administered drug preparation in bronchial mucus.

3 cl, 3 dwg, 4 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention represents a drug preparation for treating diseases caused by type 1 herpes simplex and cytomegalovirus, containing recombinant human interferon 2α, lisocyme, Licopid, carnitine 20%, vitamin E and a fatty base.

EFFECT: higher clinical effectiveness and reduced length of treatment, prolonged intercurrent periods, lower recurrent rate by prophylactic administration, reduced manifestation of neurotoxic effects of herpes viruses, lower administration of antibiotics for preventing bacterial complications in infectious-inflammatory diseases caused by herpes simplex virus and cytomegalovirus in children.

2 cl, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to medicine, namely to pharmaceutical composition, which contains enzyme deoxyribonuclease or glycyrrhizic acid or its salts: ammonium or dipotassium or trisodium glycyrrhizinate, which can be applied in medicine for treatment and prevention of viral infections, caused by DNA-containing viruses, such as herpes, shingles, human papilloma, adenoviruses, etc. In an invention claimed is a pharmaceutical composition, which includes as active ingredients - 0.001-0.5 wt % of deoxyribonuclease (DNA-ase) and 0.001-0.5 wt % of glycyrrhizic acid or its salts: ammonium or dipotassium or trisodium glycyrrhizinate, as carriers: β-cyclodextrins or lecithins 0.001-5.0 wt %, a polymer carrier 0.05-1.00 wt % and acceptable excipients.

EFFECT: obtaining the pharmaceutical composition, which contains enzyme deoxyribonuclease and glycyrrhizic acid.

3 cl, 7 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to using 1-{4-[1-(4-cyclohexyl-3-trifluoromethylbenzyloxyimino)-ethyl]-2-ethylbenzyl}azetidine-3-carboxylic acid or its pharmacologically acceptable salt in treating demyelinated peripheral neuropathy specified in chronic inflammatory demyelinated polyradiculoneuropathy, multifocal motor neuropathy with conduction block or paraproteinemic demyelinated peripheral neuropathy.

EFFECT: using 1-{4-[1-(4-cyclohexyl-3-trifluoromethylbenzyloxyimino)-ethyl]-2-ethylbenzyl}azetidine-3-carboxylic acid for treating demyelinated peripheral neuropathy .

4 dwg, 2 tbl, 2 ex

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