RU2753191C2 - New recombinant growth hormone analogue with prolonged activity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the area of biotechnology, specifically to production of recombinant human growth hormone, and can be used in medicine for treatment of diseases associated with human growth hormone deficiency. The recombinant growth hormone has a unique structure and comprises four functional areas: an area homologous to the amino acid sequence of human growth hormone, an area increasing thermal stability and functional stability of the molecule, an area providing dimerisation of the molecule, and an area providing affinity for the cell membrane.

EFFECT: invention ensures production by recombinant method in E. coli cells of human growth hormone exhibiting improved pharmacokinetics - prolonged activity and retaining biological activity of native growth hormone.

2 cl, 3 dwg, 3 ex

Description

The field of technology.

The present invention relates to long-acting human growth hormone fusion proteins, in which a physiologically active growth hormone molecule is combined with amino acid sequences that provide retardation of excretion from the body, thermal and structural stability.

Prior art.

Recombinant human growth hormone (HGH) is used to treat short stature in children (pituitary dwarfism), Turner syndrome, and hormone deficiency in adults. This therapy is of a substitutional nature, because with a deficiency of growth hormone in the body, the level of endogenous hGH decreases. Due to the fact that hGH is extremely unstable in the patient's body, therapy involves daily injections at high doses for a long time. Such therapy is extremely inconvenient for patients. Currently, an urgent task is to create prolonged-acting hGH drugs, which will lead to an improvement in the quality of life of patients and an improvement in the therapeutic effects of treatment.

HGH is eliminated from the body by two mechanisms. The first is renal clearance, in which hGH is cleared from the bloodstream by renal glomerular filtration. The renal clearance of hGH is well described in the literature and accounts for 25-53% of the total hGH clearance. The second mechanism is receptor-mediated clearance in tissues and organs, primarily in the liver, where receptor-mediated endocytosis occurs, followed by lysomal degradation of the hormone.

The pegelation method is widely used to reduce the renal clearance of pharmaceutical proteins. Pegelated hGH derivatives are not subject to glomerular filtration, but they effectively bind to the receptor and undergo endocytosis. In addition, the side effects of pegelated hGH derivatives are well known, primarily lipoatrophy at the injection site. Lipoatrophy is a medical term used to describe localized loss of adipose tissue. The ethology of lipoatrophy is presumably explained by the direct lipolytic effect of high doses of hGH. Such high concentrations occur at the injection site of viscous solutions of pegelated hGH. Most major pharmaceutical manufacturers have decided to discontinue clinical research programs for pegelated hGH derivatives.

The problems identified in the development of pegelated analogs of hGH have led to the need to find other ways to prolong the hormone in the human body. US patent 9,211,342 protects an hGH derivative that is resistant to plasma degradation. Stability is achieved by introducing at least one additional disulfide bond into the sequence of the molecule. As a result, the hormone acquires a more rigid structure that is not subject to unfolding and folding due to the thiol-disulfide exchange reaction characteristic of GH. Additionally, point mutations are introduced that reduce protein aggregation. A similar idea, but with different mutations, is described in CN 104812776. KR 20140141874 and US 9187546 protect a method for producing a hyperglycosylated hGH derivative. For this purpose, the sequence introduced the appropriate point mutations, and the hGH itself is expressed in mammalian cells capable of glycosylating the products of heterologous expression. It should be noted that the stable derivatives of hGH by themselves cannot be considered as really effective prolonged molecules. Subsequently, these molecules can be incorporated into microparticles; such a combination should give a good synergistic effect for obtaining prolonged hormone preparations.

US Pat. Nos. 9061067 and US Pat. It is known that such low molecular weight compounds have a high affinity for serum albumin. After injection, HGH derivatives with such tails interact with albumin, increasing the drug's lifetime in the blood. However, such drugs cannot get rid of receptor clearance; as a result, it is not possible to achieve a reduction in injections to one per week.

To slow down receptor-mediated excretion, several variants of hGH have been developed, in which the hormone is conjugated to large protein molecules such as albumin (TV-1106, NNC0195-0092), nonsense extended polypeptide chains VRS-317 (phase 3 clinical trials are underway), MOD- 4023 (phase 2/3 of clinical trials in progress)). This modification reduces binding to the receptor by 10-15 times. US Pat. No. 8,470,559 describes a derivative containing a GH complex and a fragment of its cytoplasmic receptor. Such a derivative has a reduced biological activity and therefore is poorly excreted by the receptor mechanism of renal clearance. Finally, US patent 9371369 protects a hGH derivative in which hGH is fused to a molecule composed of a very extended unnatural amino acid sequence. The extended fragment reduces the affinity of the GR derivative to its receptor by a factor of 20; therefore, the specific receptor clearance decreases and, accordingly, the lifespan of the GR analog in the human body increases. This can be regarded as a kind of attempt to deposit the GH molecule in the body. A decrease in the affinity for the receptor also reduces the biological activity of the drugs, which leads to an increase in the dose of the drug being administered. Increasing the dose leads to a local concentration of the active hormone, which can cause side effects on the body, for example, the already mentioned lipoatrophy. The patents CN 102875683, US 9061072 and RU 2473554 describe hGH derivatives linked to fragments of human immunoglobulin molecules. US Pat. No. 8,304,386 describes a GH derivative combined into a single molecule with a fragment of the beta chain of human chorionic gonadotropin. US Pat. No. 8,470,559 describes a derivative containing a complex of hGH and a fragment of its cytoplasmic receptor. Such a derivative has a high molecular weight and reduced affinity for membrane-bound hGH receptors and therefore is poorly cleared by the receptor mechanism of renal clearance.

There are many scientific works and patents devoted to the methods of physical depositing of native hGH. Numerous studies have shown that long-term administration of small doses throughout the day has the same biological effect as a single administration of a large dose once a day. Based on these studies, Nutropin Depot, a depot growth hormone drug, was developed and received FDA approval. This drug has been discontinued due to the complexity of the production technology (as explained by the manufacturer Genetech Inc, USA). At present, a deposited drug LB03002 (from LGLifeSciences, Korea) is being developed, which is admitted to the third phase of clinical trials. The developers of the drug use the same technology as when creating Nutropin Depot. Problems are possible for this drug, as well as for its predecessor. WO 2002030990 describes a method for the preparation of chemically crosslinked amide derivatives of hyaluronic acid, which is the main component of cartilage synovial fluid. The drug is completely compatible with the body and completely biodegradable. Based on these particles, the only currently approved long-acting GR drug has been obtained. However, this took almost 20 years, which is associated with the technical difficulty of obtaining particles of the same size, resistant to degradation for a long time and maintaining the concentration of GH at a physiologically acceptable level. US Pat. No. 8,313,767 protects a method for producing prolonged GH by precipitating a recombinant protein inside the pores of hydroxyapatite particles of a certain size. The production of hydroxyapatite particles is technologically simple and cost effective. The immobilization of GH on particles is carried out using the well-studied reaction of the hormone with zinc ions. Zinc ions stabilize well the GR dimer in the pores of the matrix and slowly release it from the surface. An important indicator of the quality of deposited GH preparations is the percentage of the hormone in a gram of microparticles. One of the highest percentages is claimed in CN 103976982. The microparticles are based on a branched PEG-polyester copolymer, the percentages being more than 80%. Other indicators of the quality of depot-GR are the particle size and viscosity of the injected drug. US patent 8729015 describes microcapsules based on lipid compositions. Lipid compositions are very flexible, small and allow the use of fine needles. Patent KR 20170043890 protects a deposition method in which a depot occurs after being introduced into a patient's body. A dextran-based polymer is used, which has the ability to harden at body temperature, capturing GH molecules in its composition. The slow breakdown of dextran leads to a slow, non-spike, increase in the concentration of GH in the blood.

The applicant has developed an analogue of hGH, combining two possibilities of prolongation - an increase in molecular weight to reduce renal filtration and deposition to reduce receptor clearance. The drug is capable, due to the unique amino acid sequence, to be deposited in the body and gradually released, and the presence of a dimerizing fragment and human theoredoxin in the structure, which increase the molecular weight, allows maintaining the necessary concentration for a long time to achieve a biological effect.

The recombinant analogue of hGH was obtained by expression in E. coli cells; methods of purification of this product have been developed. HPLC confirmed primary structure and homogeneity. The developed protein has improved pharmacokinetics and retains the biological activity of native hGH.

Recombinant hGH has a unique structure and contains three functional regions: a region homologous to the amino acid sequence of human growth hormone, a region that increases thermo-stability and functional stability of the molecule, a region that promotes dimerization, and a region that provides an affinity for the cell membrane.

The amino acid sequence of human thioredoxin was used as a fragment that improves thermal stability and functional stability. Its positive properties have been widely studied in the scientific literature.

A leucine zipper sequence was used as a dimerizing fragment. Leucine zipper is a common three-dimensional structural motif in proteins and is so named because leucines are found in every seventh position of the amino acid fragment, thereby constituting the dimerization domain of two alpha-helices. Such structures are present in both prokaryotes and eukaryotes and are one of the most important elements of protein-protein interactions.

The study of the binding of proteins to cholesterol in cell membranes has led to the establishment of sequences that interact with cholesterol in cell membranes. Consensus sequences contain the Leu / Val-X (l-5) -Tyr-X (l-5) -Arg / Lys motif. There are many membrane cholesterol recognition sequences, and any of them can be used in the proposed sequence.

The developed peptide exists in the form of a dimer, therefore it is more stable when administered and has improved pharmacokinetics and exceeds that of the native hormone by more than 500 times. The stability of the hormone in the body is also influenced by its membrane-binding region. This region provides a short-term binding of the protein to the cholesterol clusters of cell membranes, contributing to the deposition of hGH.

The technical result achieved with the implementation of this invention is to increase the effectiveness of hormonal therapy and reduce the frequency of injections during the course of treatment.

The invention is illustrated in FIG. 1-3:

FIG. 1. Chromatographic profile of the passage of the recombinant Tr2CrZhGH protein through Q-Sepharose.

FIG. 2. Chromatographic profile of elution of recombinant Tr2CrZhGH from an analytical column Kromasil 300-5С18

FIG. H. Pharmacokinetic profile of Tr2CrZhGH

Examples of implementation of the invention:

1. Obtaining a strain-producer of the recombinant protein Tr2CrZhGH.

To obtain a recombinant protein, a genetic construct was created based on the plasmid pET30a, which contains the gene SEQ ID: 2, which encodes this recombinant protein.

The plasmid was used to transfect cells of E. coli strain BL21 (DE3). The transformed cells were plated on a Petri dish with LB-agar containing 25 mg / ml of kanamycin and incubated for 16 hours at 37 ° C. Single colonies were incubated for 10 hours in 1 ml of LB containing 25 mg / ml of kanamycin. 50 μl of cell cultures were added to 0.5 ml of LB containing 25 mg / ml of kanamycin, incubated for 2 hours, and expression of the rec-MP protein was induced by adding IPTG to 1 μM. Matrix cultures were stored at -80C until the end of clone analysis. The presence of the recombinant protein was analyzed by electrophoresis in PAGE. One clone producing the largest amount of product was used as a producing strain.

2. Obtaining and chromatographic purification of Tr2CrZhGH.

The cells of the producer strain were subcultured into a culture flask with 200 ml of LB containing 25 mg / ml of kanamycin. The culture was grown on a shaker at 280 rpm at 37C until the optical density reached OD585 = 0.8E. The induction of protein expression was carried out by adding IPTG to a concentration of 1 mM. The culture was additionally cultured for 4 hours, then the cells were precipitated by centrifugation at 5000g. The cell pellet was lysed in 8M urea and processed on an ultrasonic disintegrator. The cell lysate was centrifuged at 20,000g for 2 hours. The supernatant was filtered through an SPQ-Sepharose column. The fraction that did not bind to this sorbent was chromatographed on Q-Sepharose in a NaCl gradient (starting buffer: 20 mM TrisHCl pH 8.8, 20 mM NaCl, final buffer 20 mM TrisHCl pH 8.8, 400 mM NaCl). The chromatographic profile is shown in FIG. 1. The fractions containing the target product were combined and dialyzed in 0.9% NaCl, then rechromatographed on SP-Sepharose. The purity of the product was analyzed by HPLC (Fig. 2.).

3. Study of the pharmacokinetics of the recombinant protein Tr2CrZhGH.

The study used 125 male ICR mice and 6 male NZW rabbits. Mice were injected with Tr2CrZhGH subcutaneously at three experimental doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg. The minimum dose of 1 mg / kg was administered once or weekly for 4 weeks. Doses of 5 mg / kg and 10 mg / kg were administered once. The drug was administered to rabbits once intravenously (marginal ear vein) or subcutaneously at a dose of 2 mg / kg.

The maximum concentration with a single subcutaneous injection in mice is achieved within four hours after injection, after which the concentration slowly decreased with an elimination half-life of about 63 hours. At all doses used, the drug remained in the bloodstream during the experiment (seven days). Given the long elimination half-life, the elimination time of the drug can probably be very long and potentially the drug can be detected in the blood after a long time.

The maximum concentration with subcutaneous administration of the drug to rabbits is achieved within eight hours after injection, after which the concentration slowly decreased with a half-life of about 69 hours. The maximum intravenous concentration was recorded 4 hours after injection, after which the concentration slowly decreased with an elimination half-life of about 63 hours. The clearance profile is shown in FIG. 3.

List of sequences.

<110> FGBU Institute of Biology and Development named after N.K. Koltsov RAS

<120> New recombinant analogue of prolonged-release growth hormone.

<160> NUMBER OF SEQ ID NOS: 1

<210> SEQ ID NO 1

<211> 326

<212> AA (amino acid)

<213> artificial

<400> SEQUENCE 1:

MVKQIESKTKFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSASEKYSNVIFLEDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELVGGSGGSKGGGSGGGKSGGGSGGKLLSGSSGGSGGGGSAMGSSHHHHHHRAFLEKENALLRQEVVALRQELSHLRAVLSRYQAEGGSGGGSGGSGGGSSGSGSGGKEGVLYVGSKTKSGGSGSGSGGGSFTTFTVTKYWFYRLLSALFGIPMALIWGIYFAILSFLHSGSGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

Claims (2)

1. A recombinant polypeptide having human growth hormone receptor agonist activity, characterized in that it has the amino acid sequence of SEQ ID NO: 1. 2. Pharmaceutical composition for therapy with prolonged human growth hormone based on the recombinant polypeptide of SEQ ID NO: 1 for subcutaneous, intramuscular or rectal administration.

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