JP2010138160A - Liver function protecting pharmaceutical formulation - Google Patents

Abstract

(式中、ＬＦはラクトフェリン、ＮＨＳはＮ−ヒドロキシスクシンイミジル基、ＰＯＬＹは分子量（数平均分子量）が１０，０００〜６０，０００（Ｄａ）であるポリ（アルキレングリコール）、ｎは１〜１０の整数、をそれぞれ表す）で示される分岐型非ペプチド性親水性高分子とラクトフェリンとの複合体。
【選択図】なしThe present invention provides a lactoferrin complex that is highly clinically useful as a hepatic function-protecting pharmaceutical composition with reduced antigenicity, imparted pepsin resistance, and prolonged life span in the body.
[Solution]

(Wherein LF is lactoferrin, NHS is an N-hydroxysuccinimidyl group, POLY is a poly (alkylene glycol) having a molecular weight (number average molecular weight) of 10,000 to 60,000 (Da), and n is 1 to 1 A complex of a branched non-peptidic hydrophilic polymer and lactoferrin represented by
[Selection figure] None

Description

  The present invention relates to a pharmaceutical composition for protecting liver function, comprising a complex of a branched non-peptide hydrophilic polymer and lactoferrin.

  Conventionally, a biopolymer and a non-peptide hydrophilic polymer such as polyethylene glycol (PEG) are conjugated for the purpose of adjusting the properties of the biopolymer (hereinafter referred to as “complexing”, PEG or In the case of using the similar compound, it is sometimes referred to as “PEGylation”). More specifically, complexation is generally performed by attaching an active group to the end of a non-peptidic hydrophilic polymer and reacting with a functional group present on the surface of a molecule such as a protein.

  In particular, protein and peptide complexation is important, and the antigenicity and immunogenicity are reduced by shielding the epitope by partially covering the molecular surface of the protein with a non-peptide hydrophilic polymer chain, Studies have been made on the reduction of uptake by the reticuloendothelial system and the like, and the recognition and degradation of proteolytic enzymes. Moreover, it is known that the complexed substance is delayed in the living body and the life in the body is extended. On the other hand, in complexed proteins and the like, it is frequently observed that the active site is affected by the presence of non-peptidic hydrophilic polymer and biological activity is reduced.

  Variation in the activity of individual proteins due to complexation varies from protein to protein. Furthermore, PEGylation has a non-uniform effect on multiple properties of a protein, such as PEGylation reduces in vitro antiviral activity while interferon increases antiproliferative activity in human tumor cells. Can occur. Therefore, the optimum conditions for obtaining a complex having desirable characteristics must be fully studied for each protein.

  Lactoferrin (hereinafter sometimes abbreviated as “LF”) is mainly present in mammalian milk, and is also found in neutrophils, tears, saliva, nasal discharge, bile, semen, etc., with a molecular weight of about 80,000 glycoproteins. Lactoferrin belongs to the transferrin family because it binds iron. The physiological activities of lactoferrin include antibacterial action, iron metabolism regulation action, cell proliferation activation action, hematopoiesis action, anti-inflammatory action, antioxidant action, phagocytosis action, antiviral action, bifidobacteria growth promotion action, anticancer The action, the cancer metastasis inhibitory action, the translocation inhibitory action, etc. are known. Furthermore, it has recently been clarified that lactoferrin has a lipid metabolism improving action, an analgesic / anti-stress action, and an anti-aging action. Thus, lactoferrin is a multifunctional physiologically active protein that exhibits a variety of functions, and is expected to be used for uses such as pharmaceuticals and foods to restore or enhance health. It is already on the market.

  When lactoferrin is taken orally, it is hydrolyzed by pepsin, an acidic protease present in gastric juice, and decomposed into peptides, so that lactoferrin molecules can hardly reach the intestinal tract. However, it is known that the lactoferrin receptor exists in the small intestinal mucosa in the digestive tract, and it has recently been clarified that lactoferrin is taken into the body from the intestine and expresses biological activity. Therefore, in order to exert the biological activity of lactoferrin, it is important to allow lactoferrin to reach the intestine without being hydrolyzed by pepsin in the gastric juice.

  In addition, it is known that lactoferrin is taken into hepatocytes by endocytosis via a receptor (Non-patent document 1: Suzuki et al., 2002). It has been reported that bovine lactoferrin exhibits a hepatoprotective action based on a cytokine secretion inhibitory action against a D-galactosamine (D-GaIN) / LPS-induced liver injury model (Non-Patent Document 2: Matsumoto et al., 2004). .

  Regarding lactoferrin, there is a report on a PEGylated complex (Non-patent Document 3: C. O. Beauchamp et al. Anal. Biochem. 131: 25-33 (1983)). This document describes that the complex of linear PEG and LF had a life span extended by 5 to 20 times. However, the biological activity of PEGylated LF, PEGylated There is no description about the degree of the image, uniformity, or the like.

  JP-A 2007-70277 (patent document 1) retains the iron binding ability of lactoferrin, and retains the important biological activity of lactoferrin based on the iron binding ability. A complex of a hydrophilic polymer and lactoferrin is described. This complex has resistance to proteases such as pepsin and trypsin, has a long life span in the body, can exhibit biological activity for a long time in the body, and can be formulated for further enteric treatment. The complex can reach the intestine without any treatment, and this complex has a uniform quality because it has a certain number of non-peptidic hydrophilic polymers bound to specific positions. It is also advantageous in terms of management and quality control, and is described as being particularly suitable for use as a pharmaceutical ingredient. However, it does not describe what action the complex of branched hydrophilic polymer and lactoferrin exhibits on hepatocytes.

Japanese Patent Laid-Open No. 2007-70277 Suzuki, Y. A. and Lonnerdal, B. 2002. Characterization of mammalian receptors for lactoferrin. Biochem. Cell. Biol. 80, 75-80. Matsumoto, S., Tanaka, K. 2004. Protective effect of lactoferrin against induction of fulminant hepatic failure induced by D-galactosamine and Lipopolysaccharide in rats.Milk Science, 53, 265-272. C. O. Beauchamp et al., Anal. Biochem. 131: 25-33 (1983)

  The present invention relates to a complex of a branched non-peptidic hydrophilic polymer and lactoferrin that has reduced antigenicity, is imparted with pepsin resistance, has an extended body life, and is highly clinically useful. The purpose is to provide new uses.

  The present invention has been completed by finding that lactoferrin (complex) modified with a branched non-peptide hydrophilic polymer is easily taken up by hepatocytes and exhibits an excellent liver function protecting action. is there.

That is, the present invention
[1] The following formula:

(In the formula, LF is lactoferrin, NHS is an N-hydroxysuccinimidyl group, POLY is a poly (alkylene glycol) having a molecular weight (number average molecular weight) of 10,000 to 60,000 (Da), and n is 1 to 1 Each represents an integer of 10)
A hepatic function-protecting pharmaceutical composition comprising a complex of a branched non-peptide hydrophilic polymer represented by the formula: lactoferrin;
[2] The liver function-protecting pharmaceutical composition according to [1], wherein the molecular weight of poly (alkylene glycol) in the complex with lactoferrin is approximately 40,000;
[3] The liver function-protecting pharmaceutical composition according to claim 1 or 2, wherein the complex of the branched non-peptide hydrophilic polymer and lactoferrin is more easily taken into hepatocytes than unmodified lactoferrin,
I will provide a.

  The liver plays an important role in the synthesis and storage of carbohydrates, proteins and lipids, the production of waste products, the production and secretion of bile, and the regulation of circulating blood volume, and is one of the most important organs in the human body. It is. Liver diseases have a wide variety of etiologies and pathologies, but if fatty liver is included, 30% of adults are said to have some kind of liver dysfunction. Therefore, prevention of such liver diseases and protection of hepatocytes are important.

  According to the present invention, there is provided a pharmaceutical composition for protecting hepatocytes having a remarkable effect at a dose lower than the amount of unmodified lactoferrin by using a complex of a branched non-peptide hydrophilic polymer and lactoferrin. Provided, whereby the prevention and / or treatment of such liver dysfunction can be efficiently performed. In addition, the complex lactoferrin contained as an active ingredient in the pharmaceutical composition of the present invention exhibits a therapeutic / preventive effect as compared with other therapeutic / preventive measures, while being very safe and abundant. Even if administered or ingested continuously for a long time, no side effects are exhibited. Therefore, the pharmaceutical composition of the present invention comprising a lactoferrin complex as an active ingredient is highly safe even when used for a long time.

The complex contained in the pharmaceutical composition of the present invention is:
The following formula:

(In the formula, LF is lactoferrin, NHS is an N-hydroxysuccinimidyl group, POLY is a poly (alkylene glycol) having a molecular weight (number average molecular weight) of 10,000 to 60,000 (Da) (for example, polyethylene glycol ( PEG)), n represents an integer of 1 to 10, respectively)
Indicated by

  Each POLY moiety may be linear, and may have a branched and / or pendant group.

  “Lactoferrin” (LF) used in the complex contained in the pharmaceutical composition of the present invention is natural or natural obtained from humans and various animals such as cattle, horses, pigs, sheep, goats, camels and the like. In addition to the type of lactoferrin molecule itself, it may be a functional equivalent of lactoferrin, such as a recombinant type (including a modified type in which some amino acids are substituted) lactoferrin, and an active fragment of lactoferrin. It does not matter whether it is present or its content, the species from which it is derived. Therefore, the term “lactoferrin” in the context of the present invention is used to encompass these various lactoferrins unless otherwise indicated.

  In the present invention, one or more of these lactoferrins can be appropriately selected and used. From the viewpoints of availability, economy and the like, typically, bovine-derived lactoferrin, particularly lactoferrin isolated and purified from milk is conveniently used.

  A branched non-peptidic hydrophilic polymer bonded to lactoferrin generally has a branched functional group that has a functional group capable of reacting with a functional group of lactoferrin at one end to form a covalent bond (ie, highly branched). It has two or more molecular chains) and is compatible with a living body or pharmacologically inactive. The term “non-peptidic” means that the peptide bond is not included or is not substantially included (the frequency is low enough not to affect the properties of the polymer (for example, 1 to 5 of the total number of monomer units constituting the polymer). %)).

  For example, as a branched non-peptide hydrophilic polymer,

  The following formula:

(Where NHS and POLY are the same as above)
It is possible to use the one shown in.

  Such branched non-peptidic hydrophilic polymers can be synthesized by known methods, but various types are already commercially available. The molecular weight (number average molecular weight) of the POLY moiety is generally about 500 to about 200,000, preferably about 2,000 to about 100,000, particularly preferably about 10,000 to about 60,000 (Da), most Preferably, it is about 40,000 (Da).

  The complex contained in the pharmaceutical composition of the present invention is produced by covalently bonding a branched non-peptidic hydrophilic polymer and lactoferrin by reacting each functional group by any known method. can do.

  Preferably, lactoferrin and branched non-peptidic hydrophilic polymer are added to the reaction solution at a molar ratio of 1: 1 to 1: 100. The mixing molar ratio of lactoferrin: branched non-peptide hydrophilic polymer is more preferably in the range of 1: 3 to 1:60, most preferably 1: 5 to 1:54.

  The reaction step is generally performed under conditions of pH 4 or higher, temperature 0 to 40 ° C., time 1 minute to 24 hours, preferably pH 6 or higher, temperature 4 to 40 ° C., time 10 minutes to 24 hours. That is, the pH of the reaction solution is preferably pH 6 or more, more preferably pH 6-9. Although the reaction time and the reaction temperature can be changed in close relation to each other, it is generally preferable to shorten the time when the reaction temperature is high and lengthen the time when the temperature is low. For example, when the reaction pH is around 7, under the condition that the molar ratio of lactoferrin: branched non-peptidic hydrophilic polymer is 1:10, the reaction is performed at 25 ° C. for about 1 hour, or at 16 ° C. or 4 ° C. for 24 hours. By doing so, particularly good results (such as uniform complexation) can be obtained. Further, under the condition that the molar ratio of lactoferrin: branched non-peptidic hydrophilic polymer is 1: 1, it is about 10 minutes at 25 ° C., within about 10 minutes to about 40 minutes at 16 ° C., and about 4 minutes at 4 ° C. Particularly good results are obtained with reactions within 1 hour to about 2 hours.

  The complex produced as described above is first adsorbed on a cation exchange carrier (resin) such as heparin and concentrated, and then the resulting concentrate is applied to a gel filtration carrier (resin). Can be easily purified. Specifically, for example, first, a sample containing a complex is applied to a heparin column to adsorb the complex to the column, and an eluate containing the complex that is concentrated by elution with a buffer solution with a high salt concentration is used. Gather. This eluate can then be applied to a gel filtration column for desalting and replacement with the desired buffer. If necessary, the eluate can be further concentrated appropriately by a known method such as dialysis or ultrafiltration. In another embodiment, by using a commercially available cation exchange gel filtration carrier, the two-step concentration / purification step by the cation exchange carrier treatment and the gel filtration carrier treatment can be performed in one step. It can also be done.

  Thus, the complex produced by the preferred method as described above retains the iron binding ability of lactoferrin, and therefore retains at least the important biological activity of lactoferrin based on the iron binding ability. . In addition, since it has resistance to proteases such as pepsin and trypsin through the binding of branched non-peptidic hydrophilic polymer, it has a long life in the body and can exhibit biological activity for a long time in the body. Furthermore, since it is less susceptible to digestion and degradation by pepsin in the stomach due to the complexation, it can reach the intestine sufficiently even without a pharmaceutical treatment for further enterolysis.

  In addition, the complex produced in this way has a uniform quality because a certain number of non-peptidic hydrophilic polymers are bonded to a specific position, which is advantageous in terms of production control and quality control. Particularly suitable for use as a pharmaceutical ingredient.

  The pharmaceutical composition of the present invention comprises an excipient, a disintegrating agent, a lubricant, which are routinely used in the pharmaceutical industry as a physiologically or pharmaceutically acceptable additive to the complex produced as described above. It can manufacture by mix | blending inert bases and / or additives, such as a powder agent. Various components and dosage forms that can be contained in such a pharmaceutical composition are well known to those skilled in the art.

  For example, excipients include monosaccharides or disaccharides such as lactose, sucrose, glucose, sorbitol, and lactitol, starches such as corn starch and potato starch, crystalline cellulose, and inorganic substances such as light silica gel, synthetic aluminum silicate, and metasilicate aluminum Examples include magnesium acid and calcium hydrogen phosphate. However, reducing monosaccharides and disaccharides cause an aminocarbonyl reaction with the ε-amino group of lactoferrin to denature proteins. In particular, in the presence of moisture and iron ions, the rapid aminocarbonyl reaction proceeds, so it should be avoided. Examples of the disintegrant include starches, carboxymethylcellulose (CMC), hydroxypropylcellulose (HPC), carboxymethylcellulose sodium salt, and polyvinylpyrrolidone. As the lubricant, sucrose fatty acid ester, calcium stearate, magnesium stearate and the like can be used.

  Furthermore, the pharmaceutical composition of the present invention may contain known pharmaceutically active ingredients.

  The pharmaceutical composition of this invention can be used as arbitrary dosage forms, such as a tablet, a granule, a capsule, a powder. Moreover, it can also be used as a lyophilized formulation as needed. The pharmaceutical composition of the present invention is particularly advantageous when it is in the form of an enteric preparation. Methods for producing such various preparations are well known to those skilled in the art. The term “pharmaceutical composition” in the context of the present invention includes not only those applied to humans but also those applied to animals veterinarily (veterinary medicine).

  The administration route of the pharmaceutical composition of the present invention may be any known route, and for example, any route such as oral, transdermal, injection, enteral, and rectal can be selected. Oral administration is preferred.

  The effective dosage of the pharmaceutical composition according to the present invention varies depending on the type, age, weight, physical condition, etc. of the subject to be administered, and can be administered in an amount suitable for each. When administered to a mammal, the dose can be, for example, 0.001 to 10 g / kg / day, preferably 0.01 to 5 g / kg / day. When administered to humans, the amount of active ingredient is generally 50 mg to 15,000 mg, preferably 300 mg to 6,000 mg per day. In general, the pharmaceutical composition according to the present invention can be used at a dose that is significantly smaller than the known effective lactoferrin amount (for example, 1/2 to 1/20 amount in terms of lactoferrin amount) and is used at an equivalent dose. If so, the number of administrations can be reduced.

  Such daily doses can be administered once or in portions to a subject in need of hepatocellular protection, prevention of liver disease or symptoms, treatment or improvement of the condition.

  Moreover, you may use together the pharmaceutical composition of this invention with another chemical | medical agent.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

1. Preparation of PEG-LF Bovine lactoferrin (bLF) was obtained from MG Nutritionals (Melbourne, Australia). 20 kDa branched PEG N-hydroxysuccinimidyl derivative (PEG-NHS; SUNBRIGHT GL2-200GS2) and 40 kDa branched PEG N-hydroxysuccinimidyl derivative (PEG-NHS) used as PEGylation reagents SUNBRIGHT GL2-400GS2) was purchased from NOF Corp. (Tokyo). Heparin Sepharose 6 high speed column and PD-10 desalting column were purchased from GE Healthcare (Uppsta, Sweden).

  For the PEGylation reaction, bLF and branched 20 kDa or 40 kDa PEG-NHS reagent were mixed in a phosphate buffer (PBS), pH 7.4 so that the molecular weight ratio was 1:10. The final protein concentration was 0.5 mg / ml. The reaction mixture was reacted at 25 ° C. for 1 hour and cooled to 0-4 ° C. to stop the reaction. After adjusting the pH to 7.6 with 10 mM sodium phosphate, the reaction mixture was charged into the adsorbent of the heparin sepharose 6 high speed column. Excess PEGylation reagent was removed by washing with 5 column volumes of 10 mM sodium phosphate. PEGylated lactoferrin as a reaction product (hereinafter referred to as “20 kDa-PEG-bLF” and “40 kDa-PEG-bLF”, respectively, using 20 kDa and 40 kDa branched PEG reagents) in 300 mM sodium chloride solution And unmodified bLF was eluted with 1M sodium chloride solution. Both flow rates were 2 mL / min. The eluted 20 kDa-PEG-bLF or 40 kDa-PEG-bLF was desalted using a PD-10 column and concentrated with Centriplus (trade name, CENTRIPLUS YM-50, MILLIPORE). The protein concentration was measured by Bradford protein assay using bovine serum albumin (BSA) as a standard.

2. Effect of PEGylated LF on carbon tetrachloride-induced liver injury model Carbon tetrachloride-induced liver injury model was subcutaneously administered to rats (or mice) with 5 mL / kg of carbon tetrachloride / olive oil mixture (1 mL / kg as carbon tetrachloride). It is an acute liver injury model induced by administration. Liver damage caused by carbon tetrachloride is caused by irreversible changes caused by the covalent binding of CCl ₃ (free radicals) with carbon tetrachloride (CCl ₄ ) by drug metabolizing enzymes to proteins and lipids in hepatocytes. In addition, it is said to cause degeneration and necrosis of cells by causing peroxidation of membrane lipids (Yoichi Kobayashi (1990): 14.1 Search for drugs for liver diseases, search for metabolic drugs) Drug Search III, Drug Development Volume 9 (edited by Hiroshi Saito, edited by Noriyuki Nomura), pages 227-241, Yodogawa Shoten, Tokyo). In this model, fatty degeneration and necrosis of hepatocytes were observed from 6 hours after administration, which is said to be most prominent between 24 and 36 hours after administration. In addition, it is said that the enzyme leakage of transaminase (GOT, GPT) activity and lactate dehydrogenase (LDH) activity shows the maximum value 12 to 48 hours after administration (Yobayashi, 1990, supra).

  Therefore, in this experiment, serum GOT, GPT and SOD activities were measured, histopathological analysis of liver tissue, and immunohistochemical analysis using anti-single-stranded DNA (ssDNA) antibody as the primary antibody.

  Five-week-old Wistar-Imamichi male rats were introduced from the Animal Breeding Institute (Ibaraki). Animals are housed at room temperature 22 ± 2 ° C. under a 12 hour light / dark cycle (light period 07: 00 to 19:00) and fed with standard chow diet (CE-2, Claire Japan, Tokyo) by free feeding. did. The handling and experimental methods of these animals have been approved by the Tottori University Experimental Animal Committee.

The animals were divided into 6 groups (n = 5). Group I was an untreated normal control group. Group II was a control group treated with carbon tetrachloride (CCl ₄ ) only. These two groups of animals received 30 mg / kg of bovine serum albumin (BSA) intraperitoneally.

Group III was administered intraperitoneally with 30 mg / kg of bLF (unmodified). In group IV, 20 kDa-PEG-bLF 30 mg / kg produced in the above was intraperitoneally administered. For group V, the above 1. 40 kDa-PEG-bLF 30 mg / kg produced in the above was administered intraperitoneally. Administration of these drugs (except for CCl ₄ ) was performed continuously for 3 days.

Other groups except Group I had CCl ₄ (1 mg / kg, CCl ₄ and olive oil at 1 hour after administration of bLF (unmodified), 20 kDa-PEG-bLF, or 40 kDa-PEG-bLF on the third day. : 1) was orally administered. The animals were euthanized 24 hours after CCl ₄ administration and blood and liver were collected.

<Measurement of liver deviation enzyme in serum>
The collected blood was allowed to stand at room temperature for 1 hour, and then centrifuged at 1000 × g for 10 minutes to separate serum. GPT and GOT concentrations in serum were measured by UV method (GPT or GOT assay kit, Wako, Japan). Antioxidant activity was evaluated by the nitro blue tetrazolium (NBT) reduction method using “SOD activity detection kit” (Wako, Japan).

The effect of PEG-bLF on the increase in serum GOT and GPT induced by CCl ₄ is shown in Table 1.

  Pre-administration of 40 kDa-PEG-bLF or 20 kDa-PEG-bLF significantly suppressed the increase in GOT and GPT (t-test, p <0.05). On the other hand, pre-administration of unmodified bLF slightly tended to suppress the increase in GOT or GPT, but no significant difference was observed (t-test, p> 0.05). The effects of 40 kDa-PEG-bLF and 20 kDa-PEG-bLF were also significant for unmodified bLF (t-test, p <0.05). 40 kDa-PEG-bLF had a more significant effect than 20 kDa-PEG-bLF (t-test, p <0.05).

  In addition, the increase in serum SOD activity was most remarkable in the 40k-PEG-bLF group, followed by the 20k-PEG-bLF group and the unmodified bLF group, and a significant difference was observed between the groups.

<Histopathological search>
After fixing the liver tissue with 10% (V / V) buffered formalin, hematoxylin and eosin (HE) staining was performed according to a conventional method. Pathological findings of the liver are described in French et al. (2000) (French, SW, Miyamoto, K., Ohta, Y., Geoffrion, Y., 2000. Pathogenesis of experimental alcoholic liver disease in the rat. Methods Achiev. Exp. Pathol. 13, 181-207) was scored. 1 = no more than 25% local hepatocyte damage; 2 = 25-50% local hepatocyte damage; 3 = diffuse but local Hepatocyte damage; 4 = total hepatocyte necrosis. Histological search was performed as a blind test.

Table 2 shows the histological findings of liver injury caused by CCl ₄ induction.

Injuries in the hepatocytes of the CCl ₄ treatment group were observed as steatosis and hepatocyte necrosis. Steatosis and hepatocyte necrosis were observed acutely after CCl ₄ treatment. 40 kDa-PEG-bLF and 20 kDa-PEG-bLF reduced disability scores based on steatosis and necrosis. In addition, individual differences were observed in steatosis. Histological analysis against hepatotoxicity by CCl ₄ induced, 40kDa-PEG-bLF and 20 kDa-PEG-bLF is than unmodified bLF, was shown to have a more significant inhibitory effect (t- test, p <0.05). In particular, it was remarkable with 40 kDa-PEG-bLF (t-test, p <0.05).

  Histopathologically, hepatic lobular central degeneration was mildest in the 40 kDa-PEG-bLF group, and the severity was clearly increased in the order of the 20 kDa-PEG-bLF group and the unmodified bLF group.

<Immunohistochemical analysis>
For histochemical analysis, an anti-single-stranded DNA (ssDNA) rabbit polyclonal antibody (Dakocytomation, Kyoto, Japan) was used as the primary antibody. About 1 ml of 0.01 M citrate buffer (pH 6.0) was added to the sections for ssDNA detection, and the antigen activity was removed by treatment at 95 ° C. for 5 minutes. Sections were rewaxed, dehydrated, washed with 0.05 M Tris-buffered saline (TBST; pH 7.6) supplemented with Tween, treated with 1% (V / V) hydrogen peroxide, and washed again with TBST. It was. Then, add the primary antibody, react at room temperature for 30 minutes, wash with TBST, add “Simple Stain MAX-PO (Multi)” (trade name, Nichirei, Tokyo, Japan), and react at room temperature for 30 minutes. It was. Further, after washing with TBST, color was developed with a 3,3′-diaminobenzidine solution containing 0.01% (V / V) hydrogen peroxide. After washing with TBST, counterstaining with Mayer's hematoxylin was performed.

  Microscopic examination (× 400) counted over 1,000 hepatocytes over multiple fields of view. The index of apoptosis was the percentage of the total number of hepatocytes that could eliminate nuclei (Korkolopoulou, PA, Konstantinidou, AE, Patsouris, ES, Christodoulou, PN, Thomos-Tsagli, EA, Davaris, PS 2001. Detection of apoptotic cells in archival tissue from diffuse astrocytomas using a monoclonal antibodies to single-stranded DNA. J. Pathol. 193, 377-382).

  All data are expressed as mean ± SE. The significance test between groups was performed by Student's t-test, and P <0.05 was defined as the significance level.

  The results are shown in Table 2. Pretreatment with 40 kDa-PEG-bLF or 20 kDa-PEG-bLF also significantly suppressed apoptosis (t-test, p <0.05). In particular, 40 kDa-PEG-bLF was more significantly suppressed than 20 kDa-PEG-bLF (t-test, p <0.05; Table 2).

  That is, the anti-ssDNA antibody positive rate (apoptosis%) in the hepatocytes showed the lowest value in the 40k-PEG-bLF group, followed by the 20k-PEG-bLF group and the unmodified bLF group. Differences were noted.

3. For immunohistochemical analysis experiments on PEGylated LF uptake into hepatocytes , 6-week-old Wistar-Imamichi male rats were introduced from the Animal Breeding Institute (Ibaraki). Animals are housed at room temperature 22 ± 2 ° C. under a 12 hour light / dark cycle (light period 07: 00 to 19:00) and fed with standard chow diet (CE-2, Claire Japan, Tokyo) by free feeding. did. The handling and experimental methods of these animals have been approved by the Tottori University Experimental Animal Committee.

  Under anesthesia with 25% (W / W) urethane (4 g / kg, sc), the external jugular vein was cannulated. 10 mg of 40 kDa-PEG-bLF, 20 kDa-PEG-bLF, unmodified bLF or BSA solution (dissolved in physiological saline to be 10% (W / W)) using a catheter placed in the external jugular vein / kg was administered (n = 5). Ten minutes after administration, the rats were euthanized and the livers were collected.

<Immunohistochemical analysis>
The left lobe of the liver was excised, fixed with 10% (V / V) formalin, and embedded in paraffin. 5 μm sections were prepared and immunostained with an anti-bovine lactoferrin goat polyclonal antibody (Bethyl Laboratories, Montgomery, USA).
All data are expressed as mean ± SE. The significant difference test between groups was performed by Student's t-test, and P <0.05 was defined as the significance level.

  The results of immunohistochemical analysis on PEGylated LF uptake into hepatocytes are shown in Table 3.

  By immunohistological analysis, anti-bLF antibody positive vesicles were observed in the cytoplasm of hepatocytes in all animals administered with 40 kDa-PEG-bLF, 20 kDa-PEG-bLF or unmodified bLF. There was no significant difference between the groups in the number of vesicles present per hepatocyte, but the vesicle diameters were in the order of 40 kDa-PEG-bLF> 20 kDa-PEG-bLF> unmodified bLF. Large and significant difference was observed between each group (t-test, p <0.05).

4). Effect of PEGylated LF on D-galactosamine (D-GaIN) / LPS-induced liver injury model (1)
The D-galactosamine-induced liver injury model is one of the models that can be used for evaluation of substances that improve liver function, and exhibits clear acute liver dysfunction. When D-galactosamine is administered to rats, hepatocyte necrosis is observed 6 hours after the administration, and the transaminase (GOT, GPT) activity is said to show the maximum value 18 to 24 hours after the administration. The mechanism of liver damage caused by galactosamine is not yet elucidated, but galactosamine is converted from UDP-galactosamine to UDP-glucosamine, and by capturing UTP, UTP, UDP, UMP, UDP-glucose, UDP-glucuronic acid in hepatocytes is captured. As a result of the decrease, the ability to metabolize nucleic acids, proteins and lipids is thought to be inhibited (Yubayashi, 1990, supra).

  The branched 40k-PEG-LF produced in 1 above was intraperitoneally administered at 10 mg / kg to Wistar-Imamichi rats (6 weeks old, male) for 3 days. Rats were fasted for 16 hours from the evening of the second day, and GaIN / LPS mixed solution (GaIN 400 mg / kg + LPS 50 μg / kg) was intraperitoneally administered 1 hour after the third administration of 40 k-PEG-LF, and 6 hours. Later, liver and blood were sampled, and serum GOT (IU / L) and serum GPT (IU / L) were measured.

  The results are shown in Table 4. Normal control liver enzymes were GOT 86.82 ± 3.13 IU / L and GPT 39.83 ± 4.85 IU / L.

  The 40 kDa-PEG-bLF administration group showed marked increase suppression of serum GOT and GPT. Histopathologically, inflammatory cell infiltration and hepatocyte necrosis were severe in the liver of the saline administration group, but the degree of the lesion in the liver of the 40 kDa-PEG-bLF administration group was clearly reduced. .

To summarize the above results, 40 kDa-PEG-LF is the most prominent in both hepatoprotective action and hepatocyte uptake status for the CCl ₄ -induced liver injury model, followed by 20 k-PEG-bLF group and unmodified bLF group. It has been found. Therefore, the factors that increase the hepatoprotective action due to the antioxidant action exhibited by PEGylated LF, which is a complex of branched non-peptide hydrophilic polymer and lactoferrin contained in the pharmaceutical composition of the present invention, It was speculated that an increase in the ability of PEGylated LF to be taken into hepatocytes was involved.

  LF is known to be taken into hepatocytes by endocytosis via a receptor, but this study revealed for the first time that the amount of LF taken into hepatocytes by PEGylation increases.

  For the D-GaIN / LPS-induced liver injury model, 40 kDa-PEG-bLF showed a marked effect at a lower dose than the unmodified LF amount shown in a reported example (Matsumoto et al., 2004). Therefore, the complex of branched non-peptide hydrophilic polymer and lactoferrin contained in the pharmaceutical composition of the present invention, particularly 40 kDa-PEG-bLF, enhances the hepatoprotective action based on the cytokine secretion inhibitory action of LF. It was suggested to do.

5. Effect of PEGylated LF on D-GaIN / LPS-induced liver injury model (2)
The inflammatory cytokine secretion inhibitory effect of PEGylated LF on D-GalN / LPS-induced liver injury model rats was compared with that of unmodified bLF in detail.

<Method>
Fasted rats (Wistar-Imamichi strain, male, 7 weeks old) were treated with 20 kDa-PEG-bLF, 40 kDa-PEG-bLF, unmodified bLF (20 mg / kg each), or physiological saline for 3 days, It was administered intraperitoneally. One hour after administration on the third day, a GaIN / LPS mixed solution (GaIN 600 mg / kg + LPS 200 μg / kg) was intraperitoneally administered. Sampling of blood and liver was performed after 2, 4 and 8 hours.

The experimental groups were as follows:
Saline administration group (n = 8)
Saline + GaIN / LPS administration group (n = 8)
Unmodified bLF + GaIN / LPS administration group (n = 8)
20 kDa-PEG-bLF + GaIN / LPS administration group (n = 8)
40 kDa-PEG-bLF + GaIN / LPS administration group (n = 8)

<Evaluation method>
(1) Serum aspartate transaminase (AST), serum alanine transaminase (ALT)
Serum 8 hours after administration of GaIN / LPS was used as a sample, and AST and ALT were measured using a measurement kit (trade name “Transaminase CII-Test Wako”, Wako Pure Chemicals, Osaka, Japan).
(2) Histopathological Findings of Liver Tissue Paraffin-embedded sections of liver at 8 hours after administration after administration of GaIN / LPS were prepared in the same manner as described above and observed after HE staining.
(3) Presence of apoptotic cells in liver tissue TUNEL staining was performed on paraffin-embedded sections of liver at 8 hours after GaIN / LPS administration, and morphometry software (Leica Application Suite, Leica Microsystems Ltd., Heerbrugg, Switzerland) was used. Then, the abundance of TUNEL staining positive cells per 1000 hepatocytes (apoptosis index;%) was calculated.
(4) Serum TNF-α, serum IL-6, serum IL-10, serum NO
Using serum samples 2, 4, and 8 hours after GaIN / LPS administration, using a measurement kit, TNF-α concentration (trade name “Rat TNF-α Immunoassay kit”, Cosmo Bio, Tokyo, Japan), IL-6 concentration (Trade name “Rat IL-6 Immunoassay kit” Quantikine, R & D Systems, Minneapolis, USA), IL-10 concentration (trade name “Rat IL-10 Immunoassay kit” Quantikine, R & D Systems, Minneapolis, USA), and NO concentration ( The product name “Nitric Oxide Colorimetric Assay Kit” (BIOMOL, Plymouth Meeting, PA, USA) was measured.
(5) Statistical examination Using statistical software (GraphPad PRISM, GraphPad Software, San Diego, CA, USA), conducted one-way analysis of variation (ANOVA) by Tukey's multiple comparison test (ANOVA). A significant difference was determined. A statistically significant difference was defined as P <0.05. In Tables 5 to 10 below, all numerical values are shown as mean ± SE.

<Result>
(1) Serum AST and Serum ALT
The results are shown in Table 5.

  AST and ALT were significantly elevated by GaIN / LPS treatment but were significantly suppressed by pre-administration of PEGylated bLF (Table 5). On the other hand, no significant suppression was observed by pre-administration of unmodified bLF.

(2) Histopathological findings of liver tissue Intense necrotic lesions and hemorrhagic lesions were induced by GaIN / LPS treatment. The lesion was slightly suppressed by unmodified bLF pre-administration and markedly suppressed by PEGylated bLF pre-administration.

(3) Presence of apoptotic cells in liver tissue (apoptosis index)
The results are shown in Table 6.

  In the physiological saline + GaIN / LPS administration group, the abundance of apoptotic cells in the liver tissue was high. The prevalence of apoptotic cells in the unmodified bLF pre-administration group was similar to that in the physiological saline + GaIN / LPS administration group, but cell apoptosis was reduced in the 20 kDa-PEG-bLF pre-administration group. It was remarkably suppressed by pre-administration with 40 kDa-PEG-bLF (Table 6).

(4) Serum TNF-α, serum IL-6, serum IL-10 and serum NO
The results of serum TNF-α are shown in Table 7.

  At 2 hours and 8 hours after GaIN / LPS administration, the PEGylated bLF group suppressed TNF-α production statistically significantly than the unmodified bLF administration group (Table 7).

  The results of serum IL-6 are shown in Table 8.

  At 2, 4, and 8 hours after GaIN / LPS administration, the PEGylated bLF group suppressed IL-6 production statistically significantly more than the unmodified bLF administration group (Table 8).

  The results of serum IL-10 are shown in Table 9.

  At 2 and 8 hours after GaIN / LPS administration, the PEGylated bLF group promoted IL-10 production statistically significantly more than the unmodified bLF administration group (Table 9).

  The results for serum NO are shown in Table 10.

  At 8 hours after GaIN / LPS administration, the PEGylated bLF group suppressed NO production statistically significantly more than the unmodified bLF administration group (Table 10).

  For serum TNF-α, serum IL-6, serum IL-10, and serum NO, no significant difference was observed between 20 kDa-PEG-bLF and 40 kDa-PEG-bLF.

  From the above results, it was revealed that PEGylated LF significantly and significantly suppresses the inflammation induced by GaIN / LPS more than unmodified bLF. It was suggested that suppression of TNF-α, IL-6 and NO production and enhancement of IL-10 production are greatly involved in the mechanism.

Claims (3)

The following formula:

(Wherein LF is lactoferrin, NHS is an N-hydroxysuccinimidyl group, POLY is a poly (alkylene glycol) having a molecular weight (number average molecular weight) of 10,000 to 60,000 (Da), and n is 1 to 1 Each represents an integer of 10)
A liver function-protecting pharmaceutical composition comprising a complex of a branched non-peptide hydrophilic polymer represented by the formula: lactoferrin.   The pharmaceutical composition for protecting liver function according to claim 1, wherein the molecular weight of poly (alkylene glycol) in the complex with lactoferrin is approximately 40,000.   The hepatic function-protecting pharmaceutical composition according to claim 1 or 2, wherein the complex of branched non-peptide hydrophilic polymer and lactoferrin is more easily taken into hepatocytes than unmodified lactoferrin.