JP3514924B2 - Pharmaceutical composition containing C-reactive protein - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention contains C-reactive protein (hereinafter referred to as “CRP”) as an active ingredient.
The present invention relates to a pharmaceutical composition for preventing and / or treating liver diseases (including hepatitis).

The present invention also relates to a pharmaceutical composition containing CRP as an active ingredient for preventing and / or treating a disease including hepatitis, in which an immune phenomenon contributes to lesion formation.

[0003]

2. Description of the Related Art Hepatitis and other diseases associated with reduced immune function Hepatitis is a general term for diffuse inflammatory diseases of the liver. Due to its cause, viral hepatitis mainly caused by hepatitis virus is caused, and hepatitis caused by drinking alcohol. There are alcoholic hepatitis, drug-induced hepatitis due to drugs (drug-induced liver injury), etc.

In addition, viruses other than the so-called hepatitis virus that causes only hepatitis, such as the HIV virus and Ep
Stein-Barr virus, cytomegalo
infection by virus etc., or typhoid fever, Bru
Cella, Entamoeba histolytica, and Weil's disease also present with hepatitis. Furthermore, patients whose immunological function is deteriorated due to administration of anti-cancer agents, radiotherapy, etc. are more likely to develop hepatitis.

[0005] Hepatitis is a dangerous illness accompanied by swelling of the liver, tenderness and tapping of the liver, yellow gall and the like when the inflammation is strong. Depending on the severity and illness, acute hepatitis, fulminant hepatitis, subacute hepatitis,
It is classified into persistent hepatitis and chronic hepatitis. It becomes chronic if the causative viral infection or autoimmune mechanism persists. With fulminant hepatitis and subacute hepatitis, the liver gradually shrinks. When the liver becomes chronic and is accompanied by hepatic fibrosis, the liver gradually increases in hardness. In addition, histopathologically, hepatocyte degeneration / necrosis occurs, but the degree thereof varies depending on the cause, severity, and stage of disease. Kupffer cell hyperplasia and swelling, and cell infiltration in the portal area and lobules are also seen. In addition, the findings of cholestasis such as the deposition of bile pigments and bile plugs, the regeneration of hepatocytes, and the chronic growth accompanied by the proliferation of fibers cause the liver to gradually increase in hardness. (Nanzandou Medical University Dictionary 17th Edition
(Published February 1, 1990 Nanzandou).

[0006] Despite such a potentially fatal disease, an ideal therapeutic agent for hepatitis has not yet been developed.

If the immune function is further lowered due to some circumstances when suffering from hepatitis, not only hepatitis but also various organs (gallbladder, bile duct, other digestive organs, brain,
The kidneys, lungs, etc.) may cause various complications, leading to death. Therefore, it is desired to develop a therapeutic agent that normalizes the functions of various cytokines, which are information transduction factors in immune function, and comprehensively improves the general condition.

CRP CRP is a protein present in the β-globulin fraction in serum that reacts with the capsular C-polysaccharide of Streptococcus pneumoniae, and its blood concentration is 0.2 μg due to infection, inflammation, tissue damage, etc.
It is known as a type of acute phase protein that rapidly increases from 100 / ml to several hundred times to one thousand times. CRP has a molecular weight of about 130,000 and is composed of five identical peptides, and its amino acid sequence has homology with a part of serum amyloid P protein, complement C1. It is known that the complex of CRP and C polysaccharide activates the classical pathway of complement (Biochemical Dictionary, 2nd edition, p. 615, 1990, Tokyo Kagaku Dojin). In addition, in vitro using human macrophages
Experiments in tro have shown that CRP has an IL-6-stimulating action on IL-6 production in macrophages.
Therefore, although it is presumed that CRP fulfills some important function in the immune system in the living body, the actual function in the living body is still unclear.

Although CRP is clinically known as a kind of acute phase protein, it rapidly increases in infections, sudden stress such as surgery and severe stress syndrome (produced in hepatocytes). ) Is a protein,
Used daily for the diagnosis of infectious diseases. Acute phase proteins include CRP, serum amyloid A-protein (SAA), which increases several hundred times during inflammation, ceruloplasmin, C3, etc., which increases by about 50%, and α ₁ acidic glycoprotein, α, which increases 2-4 times. ₁ There are antitrypsin, etc. (Immunology Dictionary Toshiaki Osawa et al. Ed., P. 134, November 1993 1
Published on 5th, Tokyo Kagaku Doujinshi). These proteins also have a common primary structure (eg, Ohni
shi S, Maeda S, Shimada K, Arao T, J. Biochem, 100:
849-858, 1986), which are considered to have common functions in vivo.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a safe pharmaceutical agent for preventing the onset of various types of liver diseases in humans and other animals, and for treating and / or alleviating the symptoms at various stages of liver diseases. An object is to provide a composition for use.

The present invention is also used for severe liver diseases (especially viral hepatitis) found in humans and other animals having innately or acquiredly reduced immune function, to eliminate, recover, and relieve various symptoms of hepatitis. It is intended to provide safe and / or palliative safe pharmaceutical compositions.

[0012]

The present inventors have focused on the fact that CRP is secreted in large amounts from hepatocytes in the early stage of infection of various infectious diseases. When acute phase proteins such as CRP are administered to humans or animals suffering from viral hepatitis, the metabolic functions mainly involved in the production of CRP are rested to restore the original function of the liver. , I thought it would be a therapeutic drug to normalize hepatocytes.

More specifically, in most viral hepatitis, when a virus enters the liver via blood circulation, first, a) blood vessels that feed blood to the liver (portal vein, hepatic artery).
It is captured by Kupffer cells and endothelial cells that form the inner wall of the liver sinusoid that is the terminal sinusoid (in contact with hepatocytes). Next, b) the cells that compose the sinusoidal endothelium break down, so that the virus invades the hepatocytes,
It can be interpreted as leading to the development of so-called hepatitis. Therefore, according to the present invention, administration of an acute phase protein activates Kupffer cells and endothelium that capture and phagocytose a foreign pathogenic virus and a natural resistance factor of the host (pathogen covering and complement Etc.) and the activation of the immune system can be efficiently activated while balancing the organism.

[0014] Therefore, the present inventors have found that the hepatitis virus (MH
V: Mouse hepatitis virus) or CRP in mice infected with influenza virus
It was discovered that the symptoms associated with hepatitis were prevented and ameliorated by the administration of the drug, and the present invention was completed.

In addition, CRP and other acute phase proteins indirectly act as interferons having antiviral activity, as well as acting on the hematopoietic system that promotes various infection defense functions, and activate the so-called biological defense functions. It is thought to promote efficient activation of the liquid factor (information) released by the cell group, that is, the lymphokine network. Therefore, according to the present invention, not only hepatitis but also other diseases due to immunodeficiency can be expected to have similar effects.

Therefore, the present invention is a pharmaceutical composition containing CRP as an active ingredient.

As used herein, "CRP" means a protein having the amino acid sequence of 206 amino acid residues set forth in SEQ ID NO: 1. Furthermore, one having one or more amino acid residues added, deleted or substituted in the above sequence, and one chemically or enzymatically modified, or a partial structure thereof having the above-mentioned biological activity of CRP. If so, it is included in the scope of the present invention.

The CRP may be naturally derived or derived from a recombinant protein expressed by a genetic engineering technique, but the recombinant form is advantageous in view of a pathogenic secondary infection. Natural CRP is, for example, INCST
It is available from AR (USA). For CRP, the entire base sequence of the gene has been elucidated (Woo
P, Korenberg JR, Whitehead AS, J. Biol. Chem., 260:
13384-13388, 1985), and the recombinant protein is actually expressed in Escherichia coli (Koji Uchida, Toshio Tanaka, Yasunori Kihira: Biological Sample Analysis, 17 (1):
29-30, 1994, Toshio Tanaka, Yuji Matsuo: Clinical Chemistry, 23 (supl.2): 107b, 1994). The recombinant CRP protein can be obtained based on the description in the above-mentioned literature, but can also be obtained as a commercial product from Oriental Yeast Co., Ltd. or the like.

Acute phase proteins other than CRP are also
There is something in common in the primary structure and behavior of proteins, and it is considered that they also play a common role in the living body. Therefore, these proteins may also be useful as active ingredients of the pharmaceutical composition of the present invention.

In the present specification, hepatitis refers to viral hepatitis, autoimmune hepatitis, certain cases of alcoholic hepatitis due to drinking, hepatitis due to all causes such as drug-induced hepatitis, and progressive hepatitis. Including cirrhosis. In addition, for example, hepatitis caused by a decrease in immune function caused by administration of anti-cancer agents and various drugs, or by various viral infections is also included. Further, the pharmaceutical composition of the present invention, regardless of the severity and stage of hepatitis, fulminant hepatitis,
Acute hepatitis, subacute hepatitis, persistent hepatitis, chronic active hepatitis,
It is effective against any hepatitis such as cirrhosis.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The pharmaceutical composition of the present invention comprises CRP.
Oral, rectal or parenteral (including intravenous and intracerebrospinal fluid administration) by a conventional method with a carrier used in general pharmaceutical preparations.
It is manufactured by making a formulation suitable for administration.

The active ingredient can be administered alone without being mixed with other ingredients as it is, but is administered as a pharmaceutical preparation containing the active ingredient together with a carrier or the like for the purpose of facilitating the adjustment of the dose. It is preferable.

The carrier used in these pharmaceutical preparations, although depending on the dosage form used, generally includes excipients, binders, disintegrants and the like.

Typical examples of excipients are starch, lactose,
There are white sugar, glucose, mannitol, cellulose, etc.,
Examples of the binder include polyvinylpyrrolidone, starch, sucrose, hydroxypropyl cellulose, gum arabic and the like. Examples of disintegrants include starch, agar, powdered gelatin, cellulose, CMC, etc., but other excipients, binders, and disintegrators that are commonly used may be used.

Formulations of the present invention suitable for oral administration each contain a predetermined amount of the active ingredient as tablets, capsules, powders, powders or granules, or in a non-aqueous liquid such as a syrup, emulsion or bolus. It can be provided as a suspension.

Granules are provided by uniformly mixing the active ingredient and one or more of the above-mentioned auxiliary ingredients for granulation, and sizing the mesh using a sieve. Tablets
The active ingredient can be prepared by compression or molding, optionally with one or more accessory ingredients. Capsules are produced by filling a powder or granules, in which the active ingredient is homogeneously mixed, optionally with one or more auxiliary ingredients, into suitable capsules using a filling machine or the like. Formulations for rectal administration may be provided as a suppository using a conventional carrier such as cocoa butter. Formulations for parenteral administration can be provided by sealing the active ingredient as a dry solid in a sterile nitrogen purifying container. At the time of parenteral administration, this dry solid preparation can be dispersed or dissolved in a predetermined amount of sterile water and administered to a patient.

In the production of these preparations, in addition to the active ingredient and usual auxiliary ingredients, antioxidants, buffers, flavoring agents, surfactants, thickeners, lubricants, lubricants, etc. are selected. It may further contain one or more auxiliary ingredients mentioned.

The dose of the active ingredient, CRP, will of course vary depending on the route of administration, the condition to be treated and the patient to be treated, but it is ultimately up to the judgment of the physician.

A suitable dose for treating hepatitis is 0.
The range is from 02 to 200 mg / kg (body weight) / day, and a typical preferable dose is 0.1 to 100 mg / kg (body weight).
/ Day.

The desired dosage is about 12% of the above active ingredient.
It can be administered 1-3 times or more at appropriate intervals of -24 hours.

Upon treatment, CRP may be administered as a preparation containing CRP alone as an active ingredient or may be administered as a preparation containing other active ingredients. The selection is appropriately made by a doctor according to age, symptoms and the like.

The present invention will be described in more detail according to the following examples, but the present invention is not limited to these examples.

[0033]

EXAMPLES Example 1 CRP against hepatitis virus (HV) infection
Effect of Mice In general, even when a mouse is infected with hepatitis virus, a healthy individual rarely develops it and takes the form of latent infection. However, hepatitis often develops when the immune function is lowered due to stress such as aging, administration of various drugs, and infection.

Therefore, the attenuated mouse hepatitis virus HMV
SCID mice (severe combine) in which mature lymphocytes (T cells, B cells) constituting the host's immune resistance are innately deficient or almost non-functional in infection experiments using
Hepatitis was caused in the immunodeficiency mouse), and the effect of CRP on HMV infection was examined using this SCID mouse model system.

The test virus strain MHV-T is an attenuated strain isolated by the present inventors, which was acclimated to the liver of SCID mice and infected SCID liver emulsion was used as an inoculum.
SCID mice are available at the Jackson Laboratory (Bar Ha
8-10 from Rber, Maine, USA)
It is a week-old female. 10 ³ ID ₅₀ of MHV-T was intraperitoneally inoculated per mouse. As a control, MHV-T
The same experiment was carried out for those not administered with.

As CRP, recombinant human CRP (rCRP) manufactured by Oriental Yeast Co. and purified human CRP (pCRP) manufactured by INCSTAR (US) were used. rCR
P was 1.1 mg / ml and pCRP was 2.6 mg / ml, and 5 ml of each was given 0.1 ml / mouse / dose 30 minutes before, 24 hours and 48 hours after virus infection.
After 72 hours, a total of 4 doses were intraperitoneally administered. In addition, an untreated one without CRP was used as a control (5 animals).

Autopsy was performed on the 4th day of virus infection, and the pathological and immunohistochemical histology of mice was examined according to the presence of white spots (small necrotic lesions) in liver lobes, coagulating necrotic lesions in hepatocytes, and HMV antigen in hepatocytes. Anatomical findings were performed. The results are shown in Table 1.

[0038]

[Table 1]      Table 1 Effect of CRP on SCID mice infected with attenuated MHV                                            Pathological anatomy                            Macroscopic findings     Histological findings  Immunohistological findings                  White spot of liver lobe^a)   Multiple liver^b)    Hepatocyte^c) MHV mouse (small necrotic lesion) Coagulation necrotic lesion MHV antigenInoculation number of animals I II III IV I II III I II III        rCRP 5 2 3 0 0 1 4 0 3 2 0 Inoculation pCRP 5 4 1 0 0 4 1 0 5 0 0No treatment 5 0 1 1 3 0 1 4 0 0 5        rCRP 5 5 0 0 0 5 5 0 0 5 0 0 Non-inoculation pCRP 5 5 0 0 0 5 0 0 0 5 0 0No treatment 5 5 0 0 0 5 5 0 0 5 0 0   a) I: Negative, II: 3 or more, III: 4-30, IV: 30 or more   b) I: Negative, II: Mild, III: Severe   c) I: Negative, II: Mild, III: Severe As can be seen from Table 1, CRP is caused by the MHV virus.
To improve the symptoms of mouse hepatitis caused by

Example 2 Due to influenza virus
Effect of CRP on fulminant hepatitis in mice The onset of hepatitis is also observed with pathogenic viruses other than the so-called hepatitis virus. Influenza virus (from turkey A / Turkey / UK / 1/63
/ (H7N3), a mouse liver-adapted strain, develops on the third day of infection when the mouse is infected by the nasal route of infection even with several viral amounts, and dies within 24-48 hours.
That is, fulminant hepatitis develops 4 to 5 days after the virus inoculation, and hemorrhagic bronchopulmonary inflammation and encephalitis coexist, resulting in the death of the mouse.

Fulminant hepatitis due to various viruses in humans is a disease that is extremely fatal and feared. Furthermore, in recent years, it has been found that, due to the improvement of diagnostic technology, it is not possible to actually pass through death cases due to human acute liver failure due to influenza (Yasuaki Hiromoto et al., 1995 Japanese Society for Virology (Announcement No. 1004)). , Ikuko Yui and others, same (announcement number 1003),
Atsuo Ouchi et al., Same (announcement number 1029)). Therefore, the effect of CRP was examined using an experimental system of mice infected with influenza virus.

As the influenza virus, the mouse acclimated strain derived from turkey was used. The inoculation virus is
This is a subculture strain of embryonated chicken eggs, which was cultured in the chorioallantoic fluid of infected embryonated chicken eggs. As the mouse, BALB / c mouse derived from Charles River Japan was used. The amount of virus inoculated per mouse was 300 PFU (plaque forming uni
ts) / 20 μl inoculated intranasally under deep anesthesia with a combination of xylazine, pentobarbital and teraptic.

As CRP, rCRP and pCRP were used as in Example 1. rCRP is 1.1 mg / ml,
The concentration of pCRP was 2.6 mg / ml, and 0.1 ml / mouse / dose was intraperitoneally administered to each of 5 mice, 30 times before, 24 hours, and 48 hours after the virus infection, three times in total.
In addition, an untreated one that did not receive CRP served as a control (5 animals).

On day 3 of infection, necropsy was carried out, and gross pathological findings were made for the development of hepatitis (focal necrosis of the liver and jaundice) and pneumonia. The results are shown in Table 2.

[0044]

[Table 2] BAL infected with influenza virus
Effect of CRP on B / c mice Flu testGross autopsy findings    Virus mouse pneumonia^a)  Hepatitis (focal necrotic foci of the liver and jaundice)^b) Inoculation number of animals- ＋ I II III IV               rCRP 5 5 0 0 3 2 0   Nasal inoculation pCRP 5 5 0 1 4 0 0No treatment 5 5 0 0 0 0 5   a)-: Negative +: Positive   b) I: Negative II: Mild III: Moderate IV: Severe As can be seen from Table 2, CRP is a liver caused by hepatitis virus.
Caused by influenza virus as well as flame
Severe fulminant hepatitis also improves significantly.

[0045]

[Effect] The pharmaceutical composition of the present invention significantly improves various symptoms associated with hepatitis. Furthermore, CRP, which is the active ingredient of the pharmaceutical composition of the present invention, is considered to play an important role not only in natural resistance (immunity) in the early stage of infection but also in the acquired immune response. No
There is a possibility that some effects can be expected for other diseases associated with immune disorders.

Originally, hepatocytes secrete a large amount in the early stage of infection of various infectious diseases, and supplement the acute phase proteins such as CRP, which plays a major role in natural resistance, to the pharmaceutical composition of the present invention. It is an ideal therapeutic drug that uses some of the components of the host to rescue hepatitis by helping to reconstitute the natural homeostasis of the animal.

[0047]

[Sequence list]

SEQ ID NO: 1 Sequence length: 206 amino acid residues Sequence type: Amino acid Topology: circular Sequence Type: Protein Array:   GlnThrAspMetSerArgLysAlaPheVal 10   PheProLysGluSerAspThrSerTyrVal 20   SerLeuLysAlaProLeuThrLysProLeu 30   LysAlaPheThrValCysLeuHisPheTyr 40   ThrGluLeuSerSerThrArgGlyTyrSer 50   IlePheSerTyrAlaThrLysArgGlnAsp 60   AsnGluIleLeuIlePheTrpSerLysAsp 70   IleGlyTyrSerPheThrValGlyGlySer 80   GluIleLeuPheGluValProGluValThr 90   ValAlaProValHisIleCysThrSerTrp 100   GluSerAlaSerGlyIleValGluPheTrp 110   ValAspGlyLysProArgValArgLysSer 120   LeuLysLysGlyTyrThrValGlyAlaGlu 130   AlaSerIleIleLeuGlyGlnGluGlnAsp 140   SerPheGlyGlyAsnPheGluGlySerGln 150   SerLeuValGlyAspIleGlyAsnValAsn 160   MetTrpAspPheValLeuSerProAspGlu 170   IleAsnThrIleTyrLeuGlyGlyProPhe 180   SerProAsnValLeuAsnTrpArgAlaLeu 190   LysTyrGluValGlnGlyGluValPheThr 200   LysProGlnLeuTrpPro 206

─────────────────────────────────────────────────── ─── Continuation of the front page (73) Patent holder 597126480 YC Lee Y. C. Lee, Maryland, USA 21093, Simon Court, Timonium, 1824 1824 Savo Court, Timonium, Maryland 21093, United States of America (72) Inventor Hiroshi Tamura 1346-18 Kawaguchi, Tokorozawa, Saitama Prefecture Inventor Kichitaro 3-10-1, Kofudai, Fujishiro-cho, Kitasoma-gun, Ibaraki Prefecture (72) Inventor, W Sea Lee, Maryland, USA 21093, Timonium, Sabo Court, 1824 (72) Inventor, Isamu Takagawara Mihama, Chiba City, Chiba Prefecture 4-11-1, Takasu-ku 302 (72) Yushi Matsuo 6-16-15 Senriyama Nishi, Suita City, Osaka Prefecture (72) Inventor Toshio Tanaka 580-204, Shiromachi, Nagahama City, Shiga Prefecture (56) References Special Development 63-66128 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A61K 38/00-38/58 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A pharmaceutical composition for preventing and / or treating the onset of hepatitis, which comprises C-reactive protein as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the hepatitis is viral.