阅读说明：本技术 重组蛋白用于治疗代谢疾病 (Recombinant proteins for the treatment of metabolic diseases ) 是由 黄金鼎 张婉雅 叶哲铭 于 2020-02-26 设计创作，主要内容包括：本发明揭示用于预防或治疗代谢疾病的方法及组合物,其通过使用具有如SEQ ID NO:1所述的氨基酸序列的蛋白。(Disclosed are methods and compositions for preventing or treating metabolic diseases by using a protein having an amino acid sequence as set forth in SEQ ID NO. 1.)

1. Use of a protein having the amino acid sequence as set forth in SEQ ID NO 1 for the preparation of a medicament for preventing or treating a metabolic disease in an individual.

2. The use of claim 1, wherein the metabolic disease is selected from the group consisting of hyperglycemia, impaired fasting glucose, impaired glucose tolerance, insulin resistance, hyperinsulinemia, type I diabetes, type II diabetes, refractory diabetes, and combinations thereof.

3. The use of claim 1, wherein the medicament is formulated for subcutaneous, topical, intraneural, intraperitoneal, intravenous, intramuscular, intracerebroventricular, or intrathecal administration.

4. A method for preventing or treating a metabolic disease, comprising administering to a subject in need thereof a therapeutically effective amount of a protein having the amino acid sequence of SEQ ID No. 1.

5. The method of claim 4, wherein the protein is administered subcutaneously, topically, intraneurally, intraperitoneally, intravenously, intramuscularly, intracerebroventricularly, or intrathecally.

6. The method of claim 5, wherein the protein is administered subcutaneously.

7. The method of claim 4, wherein the metabolic disease is selected from the group consisting of hyperglycemia, impaired fasting glucose, impaired glucose tolerance, insulin resistance, hyperinsulinemia, type I diabetes, type II diabetes, refractory diabetes, and combinations thereof.

8. The method of claim 4, wherein the protein is administered at a dose of 0.01 to 1mg/kg once or twice daily.

9. 1, for use in the prevention or treatment of a metabolic disease in an individual.

Background

Metabolic diseases such as type II diabetes (T2DM) and other related complications are the leading cause of death. These diseases are associated with a western lifestyle with excessive nutrient intake and lack of exercise, and are increasing worldwide. T2DM (and insulin resistance disorders) is a chronic, progressive, incompletely understood metabolic disorder, primarily characterized by hyperglycemia (hyperglycaemia) (a state of prolonged elevated blood glucose concentrations). Impaired insulin secretion, resistance to tissue action of insulin, or both, are believed to be the most common cause of T2DM pathophysiology, and T2DM is initially a series of diseases caused by tissue resistance to insulin and progresses to a state characterized by complete loss of secretory activity of pancreatic beta cells. Prolonged hyperglycemia can lead to vascular and neurological damage. Type II diabetes is highly prevalent and increasing, and is a leading cause of mortality, morbidity, and healthcare use worldwide.

There are a variety of pharmacological approaches to the treatment of T2 DM. The main classes of oral antidiabetic drugs include biguanides, sulfonylureas, phenylalanine derivatives, thiazolidinediones, TZD, dipeptidyl peptidase 4 inhibitors, sodium glucose cotransporter (SGLT2) inhibitors, and alpha-glucosidase inhibitors. However, the above methods have some side effects such as hypoglycemia (hypoglycemia) and weight gain.

Thus, there remains a need for improved therapies for metabolic diseases with fewer side effects.

Disclosure of Invention

The present inventors have unexpectedly found that a recombinant protein having an amino acid sequence as set forth in SEQ ID NO:1 (hereinafter referred to as "ES 135") is effective in treating metabolic diseases. For example, when ES135 is administered to a subject suffering from a metabolic disease, hyperglycemia, the blood glucose concentration is significantly reduced.

Accordingly, one aspect of the present invention relates to a composition for preventing or treating a metabolic disease, wherein the composition comprises ES 135. Also provided herein are methods of preventing or treating a metabolic disease comprising administering an effective amount of ES 135.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Drawings

FIG. 1 shows the hypoglycemic effects of different doses of ES135 in C57BL/6 and db/db mice.

FIG. 2 shows the effect of different doses of ES135 on lowering blood glucose in C57BL/6 and ob/ob mice.

Detailed Description

I. Introduction to the theory

Provided herein are methods and compositions for preventing or treating metabolic diseases by using ES 135. According to the diabetic mouse model study described herein, the inventors showed that ES135 was effective in lowering blood glucose concentration compared to the blank group.

Definition of

The following abbreviations are used herein:

IM (i.m.): intramuscular injection

IV (i.v.): intravenous injection

IP (i.p.): abdominal injection

SC (s.c.): subcutaneous injection

PO (p.o.): oral administration

ICV (i.c.v.): ventricular injection

IT: intrathecal injection

And (3) Bid: twice a day (twice a day)

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of particular terminology used frequently herein and are not meant to limit the scope of the present disclosure.

The articles "a" and "an" as used herein are intended to refer to one or more (i.e., to at least one) of the grammatical object of the article, unless otherwise indicated by the singular form of the article.

As used herein, the term "ES 135" refers to a recombinant protein comprising the amino acid sequence of SEQ ID NO. 1, which is disclosed in U.S. Pat. No. 7,956,033, the contents of which are incorporated herein by reference in their entirety. The amino acid sequence of SEQ ID NO. 1 is as follows: ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp are provided. In some embodiments, the sequence of ES135 is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID No. 1. In some embodiments, the amino acid sequence of SEQ ID NO. 1 has one or more modifications. For example, the amino acid sequence of SEQ ID NO:1 has N-terminal phosphoglucosation or glucuronidation as disclosed in U.S. Pat. No. 9,567,385, the contents of which are incorporated herein by reference in their entirety.

In one embodiment of the invention, the amino acid sequence of ES135 consists of the amino acid sequence of SEQ ID NO. 1.

As used herein, the terms "prevent", "preventing", or "prevention" mean to exclude, avoid, eliminate, prevent, hinder, or prevent something from happening, particularly by acting in advance.

As used herein, the terms "treatment" (therapy), "treating" (therapy), or "treatment" (therapy) refer to both therapeutic and prophylactic measures aimed at preventing or slowing (alleviating) an undesirable physiological condition, disorder, or disease, or achieving a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; narrowing the scope of conditions, disorders, or diseases; a stable (i.e., not worsening) state of the condition, disorder, or disease; delaying the onset or slowing the progression of a condition, disorder, or disease; ameliorating a condition, disorder, or disease state; and alleviation (whether partial or complete), whether detectable or undetectable, or promotion or amelioration of a condition, disorder, or disease. Treatment involves eliciting a clinically significant response without excessive side effects. Treatment also includes extending survival compared to a predetermined survival without treatment.

As used herein, the terms "therapeutically effective amount" or "effective amount" and the like refer to an amount of ES135 used in the present invention to prevent, ameliorate, or treat the symptoms associated with a disease or condition mentioned in this specification, or to provide a desired therapeutic effect. The therapeutic efficacy and toxicity of ES135 can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (which is a dose that is therapeutically effective in 50% of the population) and LD50 (which is a dose lethal to 50% of the population). The therapeutic index is the dose ratio between therapeutic and toxic effects, and it can be expressed as the ratio LD50/ED 50.

As used herein, the term "administering" when used in conjunction with a therapeutic agent means administering the therapeutic agent directly into or onto the target tissue, or administering the therapeutic agent to the patient, such that the therapeutic agent positively affects the tissue to which it is targeted. Thus, as used herein, the term "administering," when used in conjunction with a compound, peptide, or protein, can include, but is not limited to, providing a therapeutic agent in or on a target tissue; the therapeutic agent is provided systemically to the subject, such as by intravenous injection, so that the therapeutic agent reaches the target tissue. The "administration" of a composition can be achieved by oral administration, injection, topical administration, or by any method in combination with other known techniques.

As used herein, the terms "animal," "subject," "patient," and the like include, but are not limited to, human and non-human vertebrates, such as wild, non-cultured, and farm animals, preferably humans.

Specific examples of the present invention

In one aspect, the invention relates to a pharmaceutical composition for preventing or treating a metabolic disease in an individual, comprising a protein having an amino acid sequence as set forth in SEQ ID NO. 1.

In another aspect, the present invention provides a protein having an amino acid sequence as set forth in SEQ ID NO. 1 for use in preventing or treating a metabolic disease in an individual.

In yet another aspect, the present invention provides the use of a protein having the amino acid sequence as set forth in SEQ ID NO. 1 for the preparation of a medicament for preventing or treating a metabolic disease in an individual.

Metabolic disorders include, but are not limited to, hyperglycemia, impaired fasting glucose, impaired glucose tolerance, insulin resistance, hyperinsulinemia, type I diabetes, type II diabetes, refractory diabetes, and combinations thereof. In some embodiments, the metabolic disease is insulin resistance. Preferably, the metabolic disease is type II diabetes.

Hyperglycemia or hyperglycemia can be defined as fasting blood glucose concentrations above about 7, about 10, about 15, or about 20 mmol/L. In particular, hyperglycemia is defined as an individual with a fasting plasma glucose concentration above the normal range, i.e., greater than 110mg/dL (6.11 mmol/L).

Abnormal fasting glucose (IFG) is defined as the condition where the subject's fasting glucose concentration or fasting serum glucose concentration ranges from 100 to 125mg/dL (i.e., 5.6 to 6.9mmol/L), particularly greater than 110mg/dL and less than 126mg/dL (7.00 mmol/L). A "normal fasting plasma glucose" subject has a fasting plasma glucose concentration of less than 100mg/dL, i.e., less than 5.6 mmol/L.

Impaired Glucose Tolerance (IGT) is a pre-diabetic state of hyperglycemia (pre-diabetes state) defined as a two-hour glucose concentration (glycemia) of about 140 to about 199mg/dL (7.8 to 11.0mmol) in a 75 gram oral glucose tolerance test (in terms of WHO and ADA). Diabetes is considered to be due to glycemia at or above about 200 mg/dL.

Insulin resistance is defined as a state where normal amounts of insulin produce a less than normal biological response. Insulin resistance can be determined using hyperinsulinemic euglycemia clamp technique (hyperinsulinemic euglycemia clamp technique), steady state Model Assessment (HOMA), or quantitative insulin sensitivity check index (QUICKI).

Hyperinsulinemia is defined as the condition of a normal lean individual with insulin resistance with or without normotemia (euglycemia) with fasting or postprandial serum or plasma insulin concentrations rising above that of a normal lean individual without insulin resistance and a waist-to-hip ratio <1.0 (male) or <0.8 (female). A fasting serum insulin concentration greater than 25mU/L or 174pmol/L is considered hyperinsulinemia. The term "euglycemia" is defined as the condition in which the subject's fasting blood glucose concentration is within the normal range, greater than 70mg/dL (3.89mmol/L), and less than 110mg/dL (6.11 mmol/L).

Type I diabetes is caused by the inability of the body to produce insulin, and is also known as "insulin dependent diabetes mellitus" (IDDM) or "juvenile diabetes mellitus". Type II diabetes is caused by insulin resistance, and is associated with an absolute insulin deficiency, sometimes with the failure of cells to use insulin correctly. It is also known as "non-insulin dependent diabetes mellitus" (NIDDM) or "adult diabetes". Adverse effects of human tissues on insulin are believed to be associated with the insulin receptor. Diabetes is characterized by recurrent episodes or persistent hyperglycemia, and in some instances, is diagnosed by exhibiting any of the following characteristics: a. fasting serum glucose concentration is ≧ 7.0mmol/L (126 mg/dL); b. as with the glucose tolerance test, two hours after oral administration of a 75 g glucose load, plasma glucose ≧ 11.1mmol/L (200 mg/dL); c. hyperglycemia, and random plasma glucose ≧ 11.1mmol/L (200 mg/dL); d. glycated hemoglobin (Hb A1C) ≧ 6.5%.

Refractory diabetes is characterized by poor glycemic control despite appropriate treatment. In this case, even an optimal treatment regimen specifically directed to rapid symptom relief and achieving glycemic goals may not work. Many assumptions, such as poor performance in seeking health care and partial patient compliance, clinical inertia, and improper selection of treatment regimens by some physicians, as well as insufficient psychosocial support by family members or diabetes care providers, are suggestive factors for this state. As described in the literature (J diabetes. 2016Jan; 8(1):76-85), early onset age, long duration of diabetes, increased complexity and frequency of treatment, insulin use, and the presence of microvascular complications are predictors of refractory diabetes.

In another aspect, the invention relates to a method for preventing or treating a metabolic disease in a subject, comprising administering to the subject a therapeutically effective amount of a protein having an amino acid sequence as set forth in SEQ ID No. 1. The therapeutic effect of this protein has been demonstrated in animal models of diabetes, as described herein.

In some embodiments, ES135 is administered to db/db and ob/ob diabetic animal models to evaluate its hypoglycemic effects. Preferably, ES135 is administered by subcutaneous injection (s.c.). In some embodiments, ES135 is administered once, twice, three times, or less per day, such as every other day, every week, every other week, or less. As demonstrated herein, ES135 was administered once subcutaneously. The detailed study procedure is shown in the examples below.

In some embodiments, administration of ES135 normalizes blood glucose concentrations in a subject. In db/db mice, ES135 at 0.5mg/kg SC significantly reduced blood glucose concentrations (p <0.05) at 6 and 30 hours post-treatment when compared to the blank group. ES135 at 1mg/kg SC also showed statistically significant reductions in blood glucose concentrations at 6, 18, 30, and 42 hours post-treatment. However, in db/db mice over the course of the study, the combination treatment group of 0.2mg/kg ES135 with 500U/kg heparin showed a modest hypoglycemic effect (FIG. 1).

In ob/ob mice, 0.5mg/kg ES135 was administered by subcutaneous injection once, significantly reducing blood glucose concentration at 6 hours post-treatment (p <0.05) compared to the blank group. ES135 at 1mg/kg SC also showed statistically significant reductions in blood glucose concentration at 6, 18, and 30 hours post-treatment compared to the blank. However, in ob/ob mice over the course of the study, the combination treatment group of 0.2mg/kg ES135 with 500U/kg heparin showed modest hypoglycemic effects (FIG. 2).

During the study in C57BL/6 mice, one subcutaneous administration of ES135(1mg/kg) did not affect blood glucose concentration (FIGS. 1 and 2).

In some embodiments, the amount of ES135 administered is equivalent to 0.001-1mg of ES135 per kg of body weight (i.e., 0.001-1mg/kg), e.g., equivalent to 0.001-0.01, 0.01-0.05, 0.05-0.1, 0.1-0.2, 0.1-0.4, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more milligrams of ES135 per kg of body weight of the subject. As shown in fig. 1 and 2, the hypoglycemic effect of ES135 in animal models of diabetes was dose-dependent. Dose-dependent hypoglycemic effects are observed in db/db and ob/ob animal models.

Overall, treatment with ES135 significantly reduced the blood glucose assessment concentration.

According to the present invention, the ES135 may be configured in any form suitable for the selected mode of administration. Preferably, ES135 may be administered subcutaneously, topically, intraneurally, intravenously, intramuscularly, intracerebroventricularly, or intrathecally. More preferably, ES135 is administered subcutaneously.

Examples

The present invention is more specifically illustrated by the following examples. It should be noted, however, that the present invention is not limited to these examples in any way.

Hypoglycemic Effect of db/db and ob/ob mice after administration of ES135

Each group of 8 normal C57BL/6 mice and non-insulin dependent diabetes mellitus (NIDDM) male mice (ob/ob, B6.Cg-Lep < ob >/J, and db/db, C57BLKS/J Iar- + Leprdb/+ Leprdb) were administered ES135(0.2, 0.5, and 1mg/kg) once subcutaneously. All animals were allowed free access to normal laboratory food and water. In addition to normal C57BL/6 mice, ob/ob and db/db mice were used with mean blood glucose concentrations of ≧ 300mg/dL and maintained for 3-5 days prior to administration.

Blood glucose values were measured from the tail blood stream of non-fasted animals at 0 hours (prior to administration) and 6 hours, 18 hours, 30 hours, 42 hours, 54 hours and 66 hours post-ES 135 treatment with a glucometer (OptiumTM xceedTM Diabetes Monitoring System, Abbott). The percentage of serum glucose and post-treatment relative to pre-treatment group values was calculated and then a two-factor ANOVA followed by a poinferroni assay (Bonferroni test) was applied to compare the treatment groups to the blank group. P <0.05, P <0.01, and P <0.001 were considered significant differences from the blank control group.

While the foregoing written description of the invention enables one of ordinary skill to make and use what is presently considered to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples herein. Therefore, the present invention should not be limited to the specific embodiments, methods, and examples described above, but rather should be limited to all embodiments and methods within the scope and spirit of the present invention.

Sequence listing

<110> Yaxiang Shengzhi medicine GmbH

Huang Jinding

Zhang Wan ya

Leaf hucho nameplate

<120> use of recombinant proteins for the treatment of metabolic diseases

<130> EUS0006WO

<150> US 62/811,616

<151> 2019-02-28

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 135

<212> PRT

<213> Artificial sequence

<220>

<223> peptide modified from homo sapiens acidic fibroblast growth factor

<400> 1

Ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His

1 5 10 15

Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg

20 25 30

Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu

35 40 45

Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr

50 55 60

Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe

65 70 75 80

Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys

85 90 95

His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys

100 105 110

Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu

115 120 125

Pro Leu Pro Val Ser Ser Asp

130 135