Patent Application
20230107018
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “609_SeqListing_ST25.txt” created on Dec. 18, 2021 and is 57,789 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to a pharmaceutical formulation including a non-activated polypeptide TRP, and more particularly to a pharmaceutical formulation including a non-activated polypeptide TRP and a phospholipid dispersant.
A non-activated polypeptide (TRP: tissue-regenerative polypeptide) is a material having a new mechanism capable of promoting formation or regeneration of tissues such as bone, cartilage, and the like, or preventing fibrosis and hardening of organs such as the kidneys, liver, lungs, heart, and the like, and has a structure containing a PTD (protein transduction domain), which enables cell membrane permeation without the aid of a cell membrane receptor, an FAD (furin activation domain), having at least one proprotein convertase cleavage site and cleaved with the proprotein convertase to activate a non-activated TRD (tissue regeneration domain) in a cell, and a TRD, which is activated through cleavage with the proprotein convertase of FAD to promote tissue growth or formation in a cell or induce tissue regeneration (Korean Patent No. 10-00775958 and U.S. Pat. No. 8,268,590).
Non-activated TRP has a structure and properties completely different from those of conventionally known active proteins such as rhBMP or TGF-β. Specifically, all conventionally known rhBMPs and TGF-μs are biochemically activated before being introduced into human or mammalian cells, and have a three-dimensional structure (Eur. J. Biochem., 237:295, 1996; J. Mol. Biol., 287:103, 1999).
Therefore, in order to produce rhBMPs and TGF-μs, which are conventional activators, there is a problem in that it is necessary to rely on recombinant culture of mammalian animal cells such as CHO and the like, which have remarkably low productivity. Some rhBMP, such as rhBMP14 (MP-52) from BioPharm, is produced by culturing inexpensive recombinant E. coli instead of animal cells, but even in this case, there are many restrictions on the biochemical structure of the BMP to be produced. Specifically, active MP-52 may be produced using recombinant E. coli, but other active BMPs or active TGF-μs that may be administered into the human body for promoting bone or cartilage formation and regenerating various tissues, such as active rhBMP2, rhBMP4, rhBMP7, etc., are not easy to produce using recombinant E. coli, and a tertiary or quaternary structure thereof is required for pharmacological effects. Moreover, these conventional recombinant growth factors are sensitive to temperature changes, and have disadvantages of low stability at room temperature (Table 1 (comparison of differences between recombinant growth factor and TRP)).
In contrast, non-activated TRP exists in the form of a weakly soluble inclusion body, and thus has very high stability even at room temperature and is very robust even with changes in acidity (Table 1).
E. coli
However, in order to use non-activated TRP as a therapeutic agent, there has conventionally been a problem in that it is necessary to improve dispersibility through inhibition of aggregation and precipitation due to poor solubility of TRP, and to overcome low cell permeability and cytotoxicity when administered at a high dose. Moreover, when delivering a protein into a cell using PTD, the size of the protein delivery system (cargo) is very limited, and it is difficult to deliver a protein of 10 kd or more (J. Cell Sci., 129:893-897, 2016). For example, TRD and FAD in the TRP correspond to protein cargos, and BMP-2 is 44.7 kd, BMP-7 is 49.3 kd, and HGF is 83.1 kd, so intracellular permeation of the therapeutic protein using PTD is very limited. Therefore, when methods for preventing the aggregation of hydrophobic proteins and increasing the intracellular permeability of large-sized cargo are developed, it will be possible to increase the effect of TRP on treating diseases.
Accordingly, the present inventors have made great efforts to develop a new formulation for using non-activated TRP for therapeutic purposes, and thus ascertained that, when non-activated TRP is used along with a phospholipid dispersant, such as egg lecithin, soybean lecithin, triglyceride (glyceryl trioleate), LysoPC (L-α-lysophosphatidylcholine), Cremophor, or the like, (1) TRP aggregation is inhibited, (2) the cell permeability of TRP, having a large protein cargo size, is increased, (3) cytotoxicity is reduced, (4) the stability of the protein is increased, and (5) the therapeutic effect is also improved, thus culminating in the present invention.
It is an object of the present invention to provide a pharmaceutical formulation capable of ensuring dispersibility through inhibition of aggregation of non-activated TRP, increasing the stability of protein medicines, and improving cell permeability, thus increasing therapeutic efficiency.
It is another object of the present invention to provide a composition for treating fibrotic disease including TRP2 and a phospholipid dispersant.
It is still another object of the present invention to provide a method of treating or preventing fibrotic disease including administering a composition including TRP2 and a phospholipid dispersant.
It is yet another object of the present invention to provide the use of a composition including TRP2 and a phospholipid dispersant for the treatment or prevention of fibrotic disease.
It is still yet another object of the present invention to provide the use of a composition including TRP2 and a phospholipid dispersant for the manufacture of a medicament for the treatment or prevention of fibrotic disease.
In order to accomplish the above objects, the present invention provides a pharmaceutical formulation including (a) a non-activated polypeptide TRP containing a PTD (protein transduction domain), which enables cell membrane permeation without the aid of a cell membrane receptor, an FAD (furin activation domain), having at least one proprotein convertase cleavage site and cleaved with the proprotein convertase to activate a non-activated TRD (tissue regeneration domain) in a cell, and a TRD, which is activated through cleavage with the proprotein convertase of FAD to promote tissue growth or formation in a cell or induce tissue regeneration and (b) a phospholipid dispersant.
In addition, the present invention provides a composition for treating fibrotic disease including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.
In addition, the present invention provides a method of treating or preventing fibrotic disease including administering a composition including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.
In addition, the present invention provides the use of a composition including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for the treatment or prevention of fibrotic disease.
In addition, the present invention provides the use of a composition including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for the manufacture of a medicament for the treatment or prevention of fibrotic disease.
Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those typically understood by those skilled in the art to which the present invention belongs. Generally, the nomenclature used herein is well known in the art and is typical.
The non-activated polypeptide (TRP: tissue-regenerative polypeptide) of the present invention has a structure containing a PTD (protein transduction domain), which enables cell membrane permeation without the aid of a cell membrane receptor, an FAD (furin activation domain), having at least one proprotein convertase cleavage site and cleaved with the proprotein convertase to activate a non-activated TRD (tissue regeneration domain) in a cell, and a TRD, which is activated through cleavage with the proprotein convertase of FAD to promote tissue growth or formation in a cell or induce tissue regeneration, and is a material having a new mechanism capable of promoting the formation or regeneration of tissues such as bone, cartilage, and the like, or preventing fibrosis and hardening of organs such as the kidneys, liver, lungs, heart, and the like (Korean Patent No. 10-00775958 and U.S. Pat. No. 8,268,590).
Non-activated TRP exists in the form of a weakly soluble inclusion body, so it has very high stability even at room temperature and is very robust even with changes in acidity. In order to use non-activated TRP as a therapeutic agent, aggregation and precipitation due to the poor solubility of TRP should be suppressed, and low cell permeability and cytotoxicity when administered at a high dose should be overcome.
TRP is a material having a mechanism that permeates cells and is activated through refolding and processing in the cytoplasm. In order to increase the pharmacological effect thereof, it is absolutely necessary to ensure physicochemical uniformity by inhibiting aggregation of a peptide preparation and to increase the intracellular permeability of a protein having a very large molecular weight (˜50 kd) such as TRP2.
In the present invention, with the goal of developing a formulation that inhibits the aggregation of non-activated TRP, the TRP aggregation inhibitory effect of various aggregation inhibitors and dispersants was confirmed. As a result, a TRP formulation containing a phospholipid dispersant such as egg lecithin, soybean lecithin, triglyceride (glyceryl trioleate), LysoPC (L-α-lysophosphatidylcholine), Cremophor, etc. is confirmed to impart a high aggregation inhibitory effect, improve cell permeability of TRP, reduce cytotoxicity, and increase in-vivo efficacy.
Accordingly, an aspect of the present invention pertains to a pharmaceutical formulation including (a) a non-activated polypeptide TRP containing a PTD (protein transduction domain), which enables cell membrane permeation without the aid of a cell membrane receptor, an FAD (furin activation domain), having at least one proprotein convertase cleavage site and cleaved with the proprotein convertase to activate a non-activated TRD (tissue regeneration domain) in a cell, and a TRD, which is activated through cleavage with the proprotein convertase of FAD to promote tissue growth or formation in a cell or induce tissue regeneration, and (b) a phospholipid dispersant.
In the present invention, the phospholipid dispersant may be selected from the group consisting of soybean lecithin, triglyceride (glyceryl trioleate), LysoPC (L-α-lysophosphatidylcholine), and Cremophor, but is not limited thereto.
In the present invention, the lecithin may be egg lecithin, soybean lecithin, sunflower oil lecithin, canola lecithin, cottonseed lecithin, or lecithin extracted from animal fat, but is not limited thereto. Preferably, egg lecithin or soybean lecithin is used, and egg lecithin is more preferably used.
In an embodiment of the present invention, the aggregation of TRP was confirmed through DLS (dynamic light scattering, Otsuka, ELSZ-2000Zs) and confocal fluorescence microscopy. Most of the TRP particles existed at a size of 300 m or more due to aggregation, and were observed in very large aggregates, even when using a confocal microscope. Therefore, these particles exhibited low intracellular permeation efficiency and mostly tended to exist outside the cell membrane (
TRP is a material having a mechanism that permeates cells and is activated through refolding and processing in the cytoplasm. In order to increase the pharmacological effect thereof, it was confirmed that it was necessary to ensure physicochemical uniformity by inhibiting the aggregation of a peptide preparation and to increase the intracellular permeability of a protein having a very large molecular weight (˜50 kd) such as TRP2.
Since normal cells have a size of 10-20 μm, it is essential to make a formulation having a uniform size of 5 μm or less in order to use TRP for therapeutic purposes. From a pharmacological point of view for patient treatment, a formulation having a uniform size of 0.5 μm or less is preferred.
In another embodiment of the present invention, the TRP2 aggregation inhibitory effect was confirmed using sugars, amino acids, ammonium sulfate, and emulsifiers, which have conventionally been used to formulate peptides or antibodies. It was confirmed for the conventional aggregation inhibitor that the TRP aggregation inhibitory effect was insignificant (Table 2), and when 10% glycerol was used, an effect of slightly reducing the size of aggregates of TRP was confirmed. Even in this case, however, TRP was mostly present in a very large size, and TRP existed outside the cell membrane rather than permeating cells (
In another embodiment of the present invention, the TRP aggregation inhibitory effect of a TRP formulation including a dispersant such as Cremophor, egg lecithin, soybean lecithin LysoPC (lysophosphatidylcholines), glyceryl trioleate, DOTAP, DSPE-PEG-amine, and soybean oil was confirmed, and a phospholipid emulsifier such as Cremophor, egg lecithin, soybean lecithin, LysoPC, and glyceryl trioleate effectively inhibited the aggregation of TRP2 and maintained TRP2 in the form of micelles. However, it was confirmed that soybean oil, which is mainly used for weakly soluble low-molecular-weight compounds, had no aggregation inhibitory effect (Table 3 and
In another embodiment of the present invention, with regard to Cremophor, egg lecithin, soybean lecithin, LysoPC, and glyceryl trioleate, which were confirmed to exhibit an aggregation inhibitory effect, the effect of increasing cell membrane permeability of TRP was confirmed through confocal fluorescence microscopy and transmission electron microscopy. For cells administered with only TRP, TRP was mostly present on the cell membrane, but a TRP2 formulation added with an emulsifier (Cremophor, egg lecithin (EL), soybean lecithin (SL), LysoPC, and glyceryl trioleate) took a generally homogeneous form due to the reduced particle size, so it passed through the cell membrane and was thus observed in the cytoplasm (
In another embodiment of the present invention, cytotoxicity of the TRP formulation including Cremophor or egg lecithin in 293A cells was analyzed using a JuLI Stage automated cell imaging system, and when the dispersant was not used, cytotoxicity appeared with an increase in the amount of the formulation that was used (1 μg and 5 μg), and egg lecithin (EL), soybean lecithin (SL), triglyceride (glyceryl trioleate), and LysoPC did not exhibit cytotoxicity even when the amount of the formulation that was used was increased (
In another embodiment of the present invention, in order to confirm the stability of a formulation including TRP2 and egg lecithin, the formulation was frozen at −20° C. and then allowed to stand at room temperature to evaluate whether the aggregation inhibitory ability is maintained over time. TRP2 not including a dispersant rapidly aggregated within a few minutes, and the size of the aggregate increased over time. The average size of the TRP2 formulation containing egg lecithin was 150 nm or less even after 56 days, indicating that the aggregation inhibitory ability was maintained constant (
In another embodiment of the present invention, when egg lecithin was used at various concentrations and when TRP2 was used at various concentrations, it was confirmed that dispersibility was maintained depending on storage conditions and long-term storage (
In another embodiment of the present invention, when the TRP formulation including the dispersant (Cremophor, egg lecithin or soybean lecithin) was administered to an animal model, the in-vivo effect of the TRP2 formulation including the dispersant in a peritoneal dialysis (PD) animal model was confirmed. With regard to the inhibition of peritoneal fibrosis in animal models, the formulation including the dispersant showed very low ED50 compared to TRP2 not including a dispersant (Tables 5 to 9 and
The formulation of the present invention may be suitable for use in injection, and the dispersant, such as egg lecithin, soybean lecithin, triglyceride, or Cremophor, is included in an amount of 100 to 50,000 parts by weight based on 100 parts by weight of the non-activated polypeptide TRP, preferably 200 to 10,000 parts by weight based on 100 parts by weight of TRP, and more preferably 500 to 5,000 parts by weight based on 100 parts by weight of TRP.
The non-activated TRP of the present invention may be prepared in a manner in which a recombinant vector in which a nucleotide sequence encoding an FAD, a nucleotide sequence for a PTD, a nucleotide sequence for tagging, and a nucleotide sequence encoding four or more histidines for separation and purification are inserted upstream of 5′ of DNA encoding a TRD is constructed, and bacteria transformed with the recombinant vector are cultured and thus a [PTD-FAD-TRD]polypeptide is expressed, after which the culture solution is added with a urea solution, thus removing two-dimensional and three-dimensional structures of the polypeptide or transforming the polypeptide into a one-dimensional linear structure, followed by purification of the [PTD-FAD-TRD] polypeptide. For example, it may be prepared through the method disclosed in Korean Patent Application Publication No. 2006-0106606.
In the present invention, the TRD may be represented by an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 13, but the TRD is not limited thereto, so long as it is a protein having activity that promotes tissue growth or formation in cells or induces tissue regeneration. Such TRD is exemplified by a polypeptide selected from the group consisting of BMPs, TGF-β superfamily, β-NGF (β-nerve growth factor), β-amyloid, ADAMs (a disintegrin and metalloproteinase-like), TNF-α, MMPs (matrix metalloproteinases), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor, and Dickkopf.
In the present invention, the FAD may be represented by an amino acid sequence selected from the group consisting of SEQ ID NOS: 14 to 26, but the FAD is not limited thereto, so long as it has a proprotein convertase cleavage site and is cleaved with the proprotein convertase in a cell to thus activate the TRD. Also, the PTD may be selected from the group consisting of TAT, Drosophila-derived Antp peptide, VP22 peptide, and mph-1-btm, but is not limited thereto.
The PTD that is used in the present invention is TAT (YGRKKRRQRRR: SEQ ID NO: 27), but the present invention is not limited thereto. Examples of the PTD may include Drosophila-derived Antp peptide, VP22 peptide (Gene Therapy, 8:1, Blackbirch Press, 2001), mph-1-btm (US 2005/0147971), PTD-3, PTD-4 (Cancer Research, 2001, 61, 474-477), TAT48-60, Penetratin, polyarginine, CADY (FEBS Letters 2013, 587-1702), and the like. In addition, the nucleotide sequence for tagging that is used is X-press Tag, but Flag, Myc, Ha, GST, etc. may alternatively be used.
In the method of producing the non-activated TRP according to the present invention, the purification step includes binding the polypeptide to nickel-titanium beads, followed by washing with the same solution and then elution with imidazole and a high-salt buffer solution, but the present invention is not limited thereto.
In the present invention, the proprotein convertase may be furin, but is not limited thereto. Examples thereof may include PC7, PC5/6A, PC5/6B, PACE4, PC1/3, PC2, PC4, and the like.
The non-activated TRP according to the present invention does not have three-dimensional stereoregularity common to conventional active BMPs, and does not have biochemical activity by itself, but when administered in a human or mammalian body, the proprotein convertase cleavage site of FAD is cleaved with the proprotein convertase present in cells in vivo to thus activate the TRD, and the activated TRD is secreted out of the cells, thereby exhibiting the expected potency. The TRP according to the present invention preferably takes the form of a polypeptide in which PTD, FAD and TRD are fused. The TRP according to the present invention has the function of promoting the formation or regeneration of tissues such as bones, cartilage and the like in the human body, or preventing fibrosis and hardening of organs such as the kidneys, liver, lungs, heart and the like, and further induces regeneration of the original tissue.
TRPs according to the present invention may be mass-produced for practical use without being limited by the biochemical structure of proteins by culturing bacteria such as recombinant E. coli, and since the biochemically inactive state thereof is maintained before administration in vivo, the production cost thereof is only a few tenths of that of conventionally known active proteins for similar uses (rhBMPs, TGF-βs, etc.), and separation, purification, handling, storage, and administration thereof are extremely simple and convenient.
In addition, the non-activated TRP according to the present invention is a polypeptide containing a PTD, which enables cell membrane permeation without the aid of a cell membrane receptor, an FAD, having at least one proprotein convertase cleavage site and cleaved with the proprotein convertase to activate a non-activated TRD in the cell, and a TRD, which is activated in the cell through cleavage with the proprotein convertase of FAD to promote tissue growth or formation or tissue regeneration. Although the TRP has no activity by itself, it permeates organisms or cells, after which FAD is cleaved with the proprotein convertase present in a large amount in almost all cells, so the TRD is activated, and the activated protein is secreted out of the cells to thus exert the potency thereof. The TRP according to the present invention is capable of solving all problems with conventional active BMP-like proteins, which are expensive to prepare, difficult to store and handle, and essentially require a receptor.
The composition containing the non-activated TRP according to the present invention as an active ingredient is preferably prepared by mixing and diluting the active ingredient with a pharmaceutically acceptable excipient or matrix as a carrier or by enclosing the active ingredient in a carrier in a container form, depending on the administration method, dosage form, and therapeutic purpose. Moreover, the composition may be used in combination with other drugs that are beneficial for the treatment of other bone defects. Here, the preparation of a physiologically acceptable protein composition having a desired pH, isotonicity, stability, etc. may be performed using any typical method known in the art to which the present invention belongs.
In the present invention, preferable examples of the matrix include, depending on biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interface properties, biodegradable and chemical materials such as calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid or polyanhydride, biodegradable and biological materials such as bone or skin collagens, other pure proteins or cellular matrix components, non-biodegradable and chemical materials such as sintered hydroxyapatite, bioglass, aluminate or other ceramics, and combinations of the aforementioned materials, such as polylactic acid, hydroxyapatite, collagen and tricalcium phosphate. However, the present invention is not limited to the carriers described above.
In the present invention, examples of the excipient may include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, magnesium stearate, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, mineral oil, and the like.
Meanwhile, the composition containing the non-activated TRP according to the present invention is preferably used through encapsulation or injection in a viscous form for administration to a bone injury site. The dose of the composition according to the present invention is not limited because it may be adjusted in consideration of the type of excipient or carrier that is used, such as a matrix or the like, the weight of the patient's bone, the bone injury site, the condition of the injured bone, the patient's age, gender and diet, the extent of suspicion of infection, the administration time, and other clinical factors. However, for the typically known effective amount of the composition, an appropriate amount may be administered continuously or dividedly in consideration of bone weight, and additional administration may be determined while observing bone growth.
In another embodiment of the present invention, when the TRP formulation including a phospholipid dispersant (Cremophor, egg lecithin, or soybean lecithin) was administered to animal models, the in-vivo effect of the TRP2 formulation including a dispersant in a peritoneal dialysis (PD) animal model was confirmed. With regard to the inhibition of peritoneal fibrosis in animal models, the formulation including the dispersant showed very low ED50 compared to TRP2 not including a dispersant (Tables 5 to 9 and
Accordingly, another aspect of the present invention pertains to a composition for treating fibrotic disease including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.
Still another aspect of the present invention pertains to a method of treating or preventing fibrotic disease including administering a composition including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.
Yet another aspect of the present invention pertains to the use of a composition including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for the treatment or prevention of fibrotic disease.
Still yet another aspect of the present invention pertains to the use of a composition including 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for the manufacture of a medicament for the treatment or prevention of fibrotic disease.
In the present invention, the fibrotic disease may include liver fibrosis, muscle fibrosis, lung fibrosis, kidney fibrosis, cardiac fibrosis, peritoneal fibrosis, various eye diseases caused by fibrosis, and the like. This fibrosis inhibitory effect may be applied to various cancers such as liver cancer, as well as chronic diseases. Moreover, since BMP-7 inhibits TGF-beta signaling (Nature Med. 2003, 9, 964-968), it may be used in combination with an immune checkpoint anticancer drug such as a PD1/PDL-1 antibody (Nature 2018, 554, 538-543). It may also be used for various metabolic diseases, inflammation, hyperphosphatemia, and Alport syndrome.
In the present invention, the phospholipid dispersant may be selected from the group consisting of lecithin, Cremophor, triglyceride, and LysoPC (lysophosphatidylcholines), in which the lecithin may be egg lecithin or soybean lecithin.
A better understanding of the present invention may be obtained through the following examples. These examples are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.
Particularly, in the following examples, only hBMP2 and hBMP7 are exemplified as the TRD constituting the TRP, but it will be apparent to those skilled in the art that not only human-derived BMPs such as hBMP3, hBMP4, hBMP6, and hBMP14 (MP-52), but also BMPs derived from mammals such as rats, cattle, pigs and the like, and other higher animals may also be used. In addition to BMPs, it will be apparent to those skilled in the art that it is also possible to use polypeptides, such as TGF-β superfamily, β-NGF (β-nerve growth factor), β-amyloid, ADAMs (a disintegrin and metalloproteinase-like), TNF-α family including ectodysplasin-A (Eda-1), MMPs including MT1-MMP (membrane type-matrix metalloproteinase) and MMP-2, and insulin-like growth factor-1 (IGF-1).
Also, in the following examples, only the prodomain of BMP is exemplified as FAD constituting TRP, but it will be apparent to those skilled in the art that the FAD is not limited, so long as it has a proprotein convertase cleavage site and is cleaved with the proprotein convertase in the cell to activate the TRD.
Also, in the following examples, the TRP protein was dispersed using a phospholipid and ultrasound, but it will be apparent to those skilled in the art that high-pressure homogenizers or dispersers commonly used for drug manufacture may be used.
In order to confirm the aggregation of TRP, DLS (dynamic light scattering, Otsuka, ELSZ-2000Zs) and confocal fluorescence microscopy were performed.
The TRP that was used was the following TRP2 having a BMP7 sequence as TRD.
RKKRRQRRRGGLFGAIAGFIENGWEGMIDGGSTMAGTMHVRSLRAAAPH
SFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSI
LGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAV
FSTQGPPLASLQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFD
LSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGRESDL
FLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSI
NPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRSQNRSKT
PKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYA
AYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAI
SVLYFDDSSNVILKKYRNMVVRACGCHHYPYDVPDYA
Bold: PTD, Italic underlined: human BMP-7, Underlined: FAD
The particle size of TRP2 separated and purified from the inclusion body of transformed E. coli was measured using DLS. As a result, as shown in
293A cells were treated with 100 ng of this TRP2 for 4 hours, and the cells were fixed in 10% formalin, stained with an X-press monoclonal antibody (Thermo Fisher R910-25) and a secondary antibody labeled with Alexa-594 (Thermo Fisher A32744) in order to observe TRP2, and observed using a confocal microscope (Carl Zeiss, LSM700).
Based on the results thereof, very large aggregates were observed even on the low-magnification confocal microscope of
TRP is a material having a mechanism that permeates cells and is activated through refolding and processing in the cytoplasm. In order to increase the pharmacological effect thereof, it is necessary to ensure physicochemical uniformity by inhibiting the aggregation of a peptide preparation and to increase the intracellular permeability of a protein having a very large molecular weight (˜50 kd) such as TRP2.
The TRP2 aggregation inhibitory effect was evaluated using sugars, amino acids, ammonium sulfate, and emulsifiers, which have conventionally been used to formulate peptides or antibodies.
To this end, the recombinant protein TRP2 at a concentration of 1 mg/ml in PBS was diluted with various dispersants at the concentrations shown in Table 2 to make 100 μg/ml of TRP2, which was then allowed to stand at room temperature for 1 hour, followed by DLS measurement. Here, the effect of each dispersant was evaluated in a semi-quantitative manner based on the average size of stabilized particles in DLS (0: >4 μm; 1+: <4 μm; 2+: <3 μm; 3+: <2 μm; 4+: <1 μm; and 5+: 250 μm). Moreover, when it was determined that there was a dispersion effect, the stability of the particles was evaluated by additionally measuring Zeta potential.
4004 ± 236.7
2808 ± 296.8
Based on the results thereof, as shown in Table 3, it was confirmed for the aggregation inhibitor conventionally used in recombinant proteins or antibodies that the TRP aggregation inhibitory effect was insignificant, and it was confirmed for Tween 80 that there was an aggregation effect, but that the Zeta potential was very low (1.63 mV), indicative of very poor particle stability.
Moreover, 10% glycerol, which is most commonly used to prevent aggregation of recombinant proteins or antibodies, slightly reduced the aggregation size of TRP, but even in this case, TRP2 was mostly present in a very large size, from which it was found that 10% glycerol was unsuitable for use as the main dispersant of TRP2.
The TRP aggregation inhibitory effect of TRP formulations including, as an emulsifier, Cremophor EL (Merck, 238470), egg lecithin (BOC sciences, BS18J04211), soybean lecithin (TCI, L0023), LysoPC (lysophosphatidylcholines) (Avanti, 830071P), glyceryl trioleate (Sigma, T7140), DOTAP (Avanti, 890890P), DSPE-PEG-Amine (Avanti, 880128P), and soybean oil (CJ Cheiljedang) was evaluated.
To this end, the recombinant protein TRP2 at a concentration of 1 mg/ml in PBS was sonicated in a 10% glycerol solution and dispersed using various dispersants shown in Table 3, and then the formation of micelles thereof was induced using weak ultrasound. Here, the appropriate concentration of the dispersion emulsifier was determined using an emulsifier having a concentration of 0.1%. The dispersed TRP2 was allowed to stand at room temperature for 1 hour to induce aggregation, and the average size and dispersion of the particles were measured through DLS. Moreover, the aggregation inhibitory effect of these dispersants was observed using a scanning electron microscope (Carl Zeiss, model name: Merlin).
15 ± 0.2
Based on the results thereof, as shown in Table 4 and in
In addition, as shown in
Therefore, it was confirmed that the phospholipid emulsifier can be used as an aggregation inhibitor and dispersant capable of effectively dispersing TRP, unlike the conventional recombinant protein or antibody.
TRP was developed as a precursor protein prodrug of BMP-7. Therefore, in order for TRP to exhibit effective pharmacological activity, it is very important that it be effectively delivered into the cytoplasm through endosomal transport. Thus, an attempt was made to confirm that the formulation showing the excellent dispersion effect of TRP2 enhances the intracytoplasmic permeability of TRP2. With regard to Cremophor, egg lecithin, soybean lecithin, LysoPC, and glyceryl trioleate, which were confirmed to exhibit the aggregation inhibitory effect in Example 3, the effect of enhancing cell membrane permeability of TRP was evaluated through confocal fluorescence microscopy. To this end, egg lecithin (EL), soybean lecithin (SL), LysoPC, and glyceryl trioleate dispersants were prepared to a concentration of 0.1%, and Cremophor (Cre) was used at 10%. TRP2 was dispersed using each dispersant and ultrasound such that the concentration of TRP2 was 100 μg/ml, and 293A cells were treated therewith for 6 hours, fixed, and stained using an X-press monoclonal antibody (Thermo Fisher R910-25) and a secondary antibody labeled with Alexa-594 (Thermo Fisher A32744) in the same manner as in Example 1, followed by multi-layer imaging and stereoscopic analysis using a confocal microscope (Carl Zeiss, LSM700).
Based on the results thereof, as shown in
In addition, in order to directly confirm the cell permeability of the TRP protein, the protein was stained with 2.5% uranyl acetate (Electron microscopy sciences, #22400) as in Example 1, and the protein was dispersed using each dispersion formulation. 293A cells were treated with 50 ng of TRP2 contained in each dispersion formulation, embedded in a resin, and observed using an electron microscope (JEOL, JEM1011).
Based on the results thereof, as shown in
These results indicate that the TRP2 formulation containing Cremophor, egg lecithin, soybean lecithin, LysoPC, or glyceryl trioleate significantly improved the cell membrane permeability of TRP, as well as the aggregation inhibitory effect.
It was confirmed in Examples 3 and 4 that the phospholipid dispersant significantly increased the delivery of TRP2 into cells, as well as the aggregation inhibitory effect thereof. Moreover, it could be observed from the scanning electron micrographs that the TRP delivered into the cytoplasm was surrounded by a two-layered membrane structure. This suggests the possibility that efficient intracytoplasmic delivery of TRP2 using the phospholipid dispersant will utilize the endosomal pathway. To this end, the TRP protein was fluorescently labeled using an Alexa Fluor 488 (excitation: 494 nm, emission: 519 nm) protein labeling kit (Invitrogen, MP10235) according to the manufacturer's instructions. The fluorescently labeled TRP was dispersed and formed into micelles as in Example 4 using egg lecithin (TRP2-EL), soybean lecithin (TRP2-SL), glyceryl trioleate (TRP2-Glyceryl trioleate), and LysoPC (TRP2-LysoPC). Moreover, in order to detect endosomes by fluorescence, a fusion expression vector of an mCherry (excitation: 587 nm, emission: 610 nm) fluorescence gene and an endosome marker Rab7a (Ras-related protein Rab-7a) gene was inserted into pcDNA3.1 (Invitrogen) to construct an mCherry-Rab7a expression vector (SEQ ID NO: 29) containing mCherry fluorescence and human Rab7a genes.
The mCherry-Rab7a expression vector thus constructed was transfected into 293A cells to induce expression, and after 48 hours, the cells were treated for 4 hours with 100 ng of the fluorescently labeled TRP2 and observed using a confocal microscope. Here, the cell nucleus was stained with NucBlue (Invitrogen, NucBlue Live ReadyProbes, R37605).
Based on the results thereof, as shown in
It was found in Examples 1 and 4 that the water-insoluble TRP protein was present in the form of being attached to the cell membrane surface. Moreover, it was found in Example 5 that the micelles, formed using the dispersion formulation, were delivered into the cytoplasm through endosomes. Cytotoxicity occurs due to destruction of the cell membrane upon excessive administration of TRP not including the dispersant, whereas prevention of cell membrane damage through endosomal recycling becomes possible when using the dispersion formulation. Therefore, the cytotoxicity of each TRP formulation in 293A cells (Thermo Fisher, R70507) was analyzed using a JuLI Stage automated cell imaging system in order to confirm the cytotoxicity reduction effect due to the dispersant. Here, 5000 293A cells were spread in a 24-well plate, and the cells were allowed to adhere for 16 hours. Thereafter, the cells were treated with TRP2 dispersed in various forms at various concentrations, and cell proliferation was observed using a real-time microscope mounted on a JuLI Stage to analyze cytotoxicity.
Based on the results thereof, as shown in
In protein medicines, protein stability is a very important issue affecting reproducibility, approval, safety, and drug efficacy. In order to confirm the stability of conventional TRP2, it was frozen at −20° C. and allowed to stand at room temperature to evaluate whether the aggregation inhibitory ability was maintained over time. As a result, protein aggregation occurred very quickly (within 10 minutes) due to the aggregation effect depending on the hydrophobic physicochemical properties of TRP, described in Table 1 (
In order to evaluate the effect of the dispersion formulation on protein stability in addition to the dispersion formulation effect described in Examples 3 to 5, TRP2 (TRP2-EL) was dispersed using 0.1% egg lecithin, and the stability of the protein drug was measured through DLS over time.
Based on the results thereof, as shown in
Storage temperature has a great influence on the stability of protein medicines. In order to confirm this influence, TRP2 was dispersed at a concentration of 100 μg/ml using 0.1% egg lecithin, and the stability thereof depending on various storage temperatures and periods was measured.
Based on the results thereof, as shown in
In order to confirm the TRP2 stability of dispersants other than egg lecithin, TRP2 was dispersed using 10% Cremophor, 0.1% soybean lecithin (TRP2-SL), 0.5% glyceryl trioleate (TRP2-Glyceryl trioleate), and 0.5% LysoPC (TRP2-LysoPC), and the stability of the particles was measured through DLS immediately after dispersion (Day 0) and after storage in a refrigerated state at 4° C. for 30 days and 70 days.
Based on the results thereof, as shown in
Egg lecithin is used at various concentrations in medicines. In order to confirm the stability of the formulation depending on the concentration of egg lecithin, TRP2 was dispersed at a concentration of 100 μg/ml using 0.5% egg lecithin, and the stability of the protein particles depending on various storage temperatures and periods was measured through DLS.
Based on the results thereof, as shown in
In addition, in order to confirm the aggregation inhibitory effect depending on the dose of protein medicine, TRP2 was dispersed at various concentrations of 100 to 500 μg/ml using 0.1% egg lecithin and stored in a refrigerated state at 4° C. for various periods, and the egg lecithin dispersibility depending on the TRP concentration was measured.
Based on the results thereof, as shown in
In order to evaluate whether the cell permeability of TRP is maintained even after long-term storage using the dispersant, 293A cells were treated for 4 hours with TRP2 dispersed using 0.1% egg lecithin and stored in a refrigerated state for 108 days, followed by observation using a confocal microscope.
Based on the results thereof, as shown in
Based on the results thereof, as shown in
A change in the therapeutic effect of the TRP formulation including a dispersant (Cremophor, egg lecithin or soybean lecithin) was confirmed when the same was administered to an animal model. In the above examples, effects of increasing the dispersibility of TRP2 protein including the phospholipid such as egg lecithin, Cremophor, soybean lecithin, glyceryl trioleate, and LysoPC, improving safety, enhancing the drug efficacy due to an increase in cell permeation efficiency, and improving stability were confirmed. A peritoneal fibrosis model was used to confirm enhancement of the therapeutic effect based on these effects.
For animal experiments, a peritoneal dialysis catheter (Access technologies, Rat-O-Port, 7 fr×24″ silicone catheter with round tip) was inserted into the abdominal cavity of male Sprague-Dawley rats weighing 250-280 g. Every day from one week after surgery, the control group was administered with 20 ml of a saline, and the experimental group was administered with a peritoneal dialysis fluid including 4.25% high-concentration glucose. In order to confirm the effect of the TRP2 formulation on treating peritoneal fibrosis, various doses of TRP2 were mixed with the peritoneal dialysis fluid and injected. As such, the TRP2 protein drug was administered once a week. Four weeks after administration of the peritoneal dialysis fluid, each animal was sacrificed, and the peritoneum was collected, fixed in 10% neutral formalin, embedded in paraffin, and stained with Masson's Trichrome, and thus the extent of histological fibrosis was determined by measuring the fibrosis thickness of at least 30 sites.
In the case of conventional undispersed TRP2, as shown in Table 5 and in
In order to confirm the therapeutic effect using a dispersant such as Cremophor, egg lecithin, or soybean lecithin, TRP2 was dispersed using 10% Cremophor, 0.1% egg lecithin, and 0.1% soybean lecithin, and various doses thereof were mixed with the peritoneal dialysis fluid and administered once a week.
Based on the results thereof, as shown in Tables 6 to 8 below and in
When the pharmaceutical formulation according to the present invention is used, dispersibility is improved through inhibition of aggregation of non-activated TRP, cell permeability is increased, cytotoxicity is reduced, stability of protein medicines is increased, and therapeutic effects are also improved.
Although specific embodiments of the present invention have been disclosed in detail as described above, it will be obvious to those of ordinary skill in the art that the description is merely of preferable exemplary embodiments, and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
An electronic file is attached.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0076443 | Jun 2019 | KR | national |
This is a United States national phase under 35 USC § 371 of International Patent Application No. PCT/KR2020/007011 filed May 29, 2020, which in turn claims priority under 35 USC § 119 of Korean Patent Application No. 10-2019-0076443 filed Jun. 26, 2019. The disclosures of all such applications are hereby incorporated herein by reference in their respective entireties, for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/007011 | 5/29/2020 | WO |
