JPH1017476A - Parenteral pharmaceutical preparation for sepsis and prevention and treatment with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject pharmaceutical preparation, usable for preventing and treating sepsis and capable of preventing the onset of the sepsis or remarkably alleviating the septic morbid state such as multiple organ failure by suspending a phospholipid therein. SOLUTION: This parenteral pharmaceutical preparation for sepsis is obtained by suspending a phospholipid (preferably an egg yolk lecithin and/or a soybean lecithin) preferably at 0.01-15% concentration therein. When intravenously administered, the phospholipid mobilizes a lipoprotein in vivo and the lipoprotein is capable of inactivating an endotoxin and/or an endotoxin-polysaccharide- bonded protein conjugate. Furthermore, the mobilized lipoprotein is preferably the one other than the lipoprotein-X (Lp-X). The pharmaceutical preparation is stable even by blending and mixing thereof with a nourishing composition used for supplementing nutrition and can stably be administered through an administration route for nutrients.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a parenteral preparation comprising a phospholipid suspension used for prevention and treatment of sepsis, and a method for prevention and treatment using the same.

[0002]

2. Description of the Related Art For reference, see "Biochemistry and Application of Physiologically Active Lipids", Koshobo, 1993, Chapter 5, pp. 85-119.
Phospholipids are a kind of complex lipids containing phosphoric acid in the form of monoester or diester, and are described as being classified into phospholipids and phosphonolipids.Phospholipids are components of biological membranes in living organisms, and phospholipids It is also a precursor of bioactive substances such as prostaglandins composed of metabolites.

[0003] Conventionally, lecithin containing diacyl phospholipid as a main component has been used as several kinds of pharmaceuticals. For example, pulmonary surfactant is suspended in physiological saline for treatment of respiratory distress syndrome. Administered intravascularly, polyenephosphatidylcholine is used as an oral capsule to improve liver function in chronic liver disease, treat fatty liver and hyperlipidemia, and soy lecithin is used as a granule in hyperlipidemia. ing. In addition, yolk lecithin as an emulsifier has been reported to be used in fat emulsions,
No pharmaceutical use for sepsis is disclosed.

JP-A-59-44326 discloses that a pharmaceutical product containing lecithin and / or cholesterol is treated by a surface active agent layer to adsorb microorganisms to cells by contact between the microorganisms and an adhesion receptor. Hinder,
It is disclosed that the action of peeling off the adsorbed microorganisms prevents the toxins of the microorganisms from reaching the cells and interrupts the growth of the microorganisms.However, the effect of inactivating endotoxin caused by the microorganisms is disclosed. Not disclosed.

[0005] JP-A-6-24973 discloses a pharmaceutical use of a fat emulsion containing a medium-chain fatty acid triglyceride for sepsis, wherein egg yolk phospholipid and / or soybean phospholipid is used as an emulsifier for the fat emulsion. However, this method is administration to sepsis by medium-chain fat administration. The preparation of commonly known fat emulsions requires a complicated process, and currently commercially available soybean oil fat emulsions can be used in patients with thrombosis, patients with severe liver or blood clotting disorders, It is not usually administered to patients with hematosis or diabetic patients with ketosis, and if administered, it must be administered slowly. In many cases, septic patients are accompanied by hepatic injury, enhanced intravascular coagulation, or multiple organ failure. Therefore, it is not practical to use fat emulsions for septic patients, since it must be done carefully.

The Japan Society of Foreign Affairs, Vol. 92, No. 12, 1990 p1
678-1684 describe that lipoprotein X (Lp-X) is produced by large dose administration of 10% soybean oil fat emulsion (intralipid) using egg yolk lecithin as an emulsifier. However, this is an increase in abnormal lipoprotein called lipoprotein X in the LDL fraction, and the fraction by centrifugation only appears in the LDL fraction. This lipoprotein X is electrophoresed on the cathode side in the analysis by electrophoresis, and it has not been reported that Lp-X inactivates endotoxin.

JP-T 8-500117 discloses that to reduce the toxicity caused by endotoxin, which is considered to be the cause of sepsis, it is necessary to use (i) a non-apolipoprotein peptide and (ii) a lipid associated with a toxic substance produced by endotoxin. Methods of administering the containing particles are disclosed.
Also, using a non-apolipoprotein peptide as a raw material, a method of using one made by surfactant dialysis together with phosphatidylcholine, a method of administering one of the non-apolipoprotein peptide or lipid, and then administering the other.
A method for administering a lipid associated with endotoxin to a patient having a high apolipoprotein blood level, a method for administering a lipid-binding peptide to a hyperlipidemic patient, and the like are described.

[0008] The preparation of these compounds is not technically easy, and although the pharmacological effect of containing a peptide is recognized, it is difficult to develop the drug as a pharmaceutical by solving the difficulties of the preparation, Need to be removed. Furthermore, even if a method using a peptide or lipid that is not an apolipoprotein alone is theoretically possible, the target patients are limited. As described above, lipids have not been sufficiently studied, the target patients are limited, and there is still room for improvement.

Infect. Immun. Vol. 61, 1993 p.
5140-5146 describes that the interaction between endotoxin and human lipoprotein suppresses endotoxin-induced activation of human monocytes and release of cytokines (monokines). LPS (lipopolys
accharide) is found in lipoprotein fractions and apolipoprotein fractions by ultracentrifugation, and inactivation of endotoxin by lipoproteins is due to LDL (low density lipopr
otein) is stronger than HDL (high density lipoprotein) and VLDL (very low density lipoprotein).
) Does not inactivate endotoxin, and lipids are not necessarily required and purified apolipoprotein A-
No. 1 (apolipoprotein abundant in the HDL fraction) was found to be found to suppress LPS-induced monokine release to the same extent as HDL in the absence of physiological lipids.

[0010] Purification of LDL requires complicated production steps such as ultracentrifugation, and it is not realistic to produce LDL in large quantities as a medicament. It is considered that the virus may be contaminated, and even if it is from animals other than humans, the problem of antigenicity due to heterologous proteins and the transmission of prions and the like, which are considered to be the cause of mad cow disease, are also not preferable. further,
Apolipoprotein B in the apolipoprotein fraction present in the LDL fraction cannot be used in the absence of a surfactant because it does not dissolve in water, and attempts to inactivate endotoxin using LDL are difficult for the above reasons. Met.

[0011]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the problems in using such a lipoprotein of the prior art in pharmaceutical preparations. Have sufficient safety to use for the purpose,
It is an object of the present invention to provide a parenteral preparation for sepsis that can be actually used as a medicament and a novel method for preventing and treating sepsis.

[0012]

Means for Solving the Problems In order to achieve the above object, the present inventors have noticed that lipoprotein inactivates endotoxin and / or endotoxin-lipopolysaccharide-binding protein complex, and particularly, preparation of a pharmaceutical preparation. As a result of intensive research on a complete and inexpensive pharmaceutical composition that is easy to produce, can be mass-produced, and free from viruses and the like, it has been found that a substance capable of producing lipoproteins such as LDL in vivo can be found. If possible, it was concluded that endotoxin and / or endotoxin-lipopolysaccharide-binding protein complex could be inactivated and used as a pharmaceutical composition, and the present invention was completed.

[0013] That is, the present invention relates to a preparation comprising a suspension of a phospholipid, wherein when administered intravenously, the phospholipid mobilizes lipoprotein in vivo and the lipoprotein is converted to endotoxin and / or endotoxin. A parenteral preparation for sepsis characterized by inactivating a lipopolysaccharide-binding protein complex.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION As the phospholipid used in the present invention, any phospholipid that can be administered intravenously for pharmaceutical use can be used, for example, pulmonary surfactant,
Examples thereof include polyene phosphatidylcholine, soybean lecithin, egg yolk lecithin, and synthetic lecithin. The phospholipid used in the present invention is preferably at least one selected from egg yolk lecithin, soybean lecithin, and synthetic lecithin, and is currently used as an emulsifier for fat emulsions, and its safety to living organisms has been confirmed. Egg yolk lecithin and / or soy lecithin are most preferred.

The method for suspending the phospholipid in the present invention can use all methods known to those skilled in the art, and can be prepared by any method such as ultrasonic treatment, a high-pressure injection emulsifier, and a homogenizer. . In the present invention, a phospholipid is added to an aqueous solution, suspended by a homogenizer, and further prepared by an ultrasonic emulsifier and / or a high-pressure jet emulsifier so as to have a desired particle type.

The concentration of the phospholipid in the suspension is not particularly limited as long as it can be adjusted, but is preferably 0.01 to 15%.
% By weight. If the concentration exceeds 15%, it is difficult to prepare a suspension having a uniform particle size, and the viscosity becomes difficult to be administered intravenously. If the concentration is less than 0.01%, the therapeutic effect is insufficient, which is not preferable. . When the phospholipid is used in a mixture with another nutritional composition, it can be mixed or used in advance in consideration of the total amount of the administered liquid.

The particle diameter of the phospholipid suspension of the present invention is not particularly limited as long as it can be adjusted, but from the viewpoint of the stability of the suspension, the particle diameter is preferably 400 nm or less. Those having a thickness of 200 nm or less are preferred.

The phospholipid suspension of the present invention can be administered alone or as a mixture with a nutritional composition. The nutrient composition used as a mixture may be of any composition as long as it is generally used by those skilled in the art, but is preferably at least one nutrient composition selected from amino acids, sugars, electrolytes, and vitamins. The phospholipid suspension can be previously mixed with the nutritional composition or administered simultaneously. The phospholipid suspension of the present invention can be administered to a septic patient complicated with liver injury because it does not contain neutral fat. In addition, the suspension obtained in the present invention can be freeze-dried, and can be used by dissolving it in physiological saline, water for injection, or the like, if necessary. Further, the present invention can be used in the form of a liposome preparation of a phospholipid or the like.

The term "sepsis" for the purpose of prevention and treatment according to the present invention refers to a systemic infection caused by the invasion of bacteria into the circulatory system, and includes those caused by either Gram-positive bacteria or Gram-negative bacteria. In particular, it includes endotoxemia in severe septic multi-organ failure complicated by severe symptoms of endotoxemia due to Gram-negative bacteria, septic shock and multiple organ failure. By administering the suspension of the phospholipid of the present invention to such septic patients for the purpose of inactivating endotoxin and / or endotoxin-lipopolysaccharide binding protein complex, bacteremia during sepsis can be improved. Or septic shock or proteolytic enzyme elastase, which inhibits the activation of monocytes and macrophages by endotoxin, and is thought to be caused by systemic inflammatory reactions caused by induction of monokines, cytokines, etc., or tissue damage by free radicals. Obstacles and the like can be suppressed. It is also possible to prevent septic multiple organ failure mainly caused by endotoxin, and the method of the present invention is a very useful prevention and treatment method.

The lipoprotein mobilized in the present invention is:
It is a lipoprotein that is not abnormal lipoprotein X (Lp-X).
L, LDL and chylomicron. Here, lipoprotein X is an abnormal lipoprotein (lipoprotein-X: Lp-X) found in the plasma of a patient in cholestatic disease and named by Seidel et al. It is regarded as important and has the feature of being electrophoresed on the cathode side in electrophoresis (clinical examination
Vol. 29, No. 11, p1383-1386, 198
5). In the present invention, it is preferable that the lipoproteins of the HDL and LDL fractions are recruited, and in particular, the LDL fraction is recruited. The lipoprotein mobilized in vivo has an effect of inactivating endotoxin and / or endotoxin-lipopolysaccharide-binding protein complex.

The term "inactivation of endotoxin" as used in the present invention refers to the suppression of endotoxin-induced sepsis by parenteral administration of phospholipids, and the death, tissue damage, and shock caused by endotoxin-induced sepsis. Say to prevent. The endotoxin-lipopolysaccharide-binding protein complex refers to a complex of endotoxin and lipopolysaccharide-binding protein, and it is known that endotoxin forms the complex and significantly enhances macrophage activation. Further, the lipopolysaccharide-binding protein is widely known as a protein that specifically binds to endotoxin, and it is a known fact that it binds to endotoxin.

The present invention provides a method for administering lipoprotein X by administering a phospholipid.
It is characterized by the recruitment of lipoproteins other than (Lp-X), but is not limited to this action. By administering the phospholipid suspension of the present invention, the mortality of the sepsis model has been remarkably improved, and not only the mobilization of lipoproteins in phospholipids but also the direct action of phospholipids, It applies to all of the preventive and therapeutic effects of phospholipids.

[0023]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Suspension Example 1 12 g of egg yolk lecithin as a phospholipid was mixed with 950 ml of an aqueous glycerin solution at about 90 ° C., and mixed with a homogenizer to form a suspension of about 5 g.
After pre-suspension for 1 minute, a suspension having a uniform particle size was obtained with a high-pressure injection emulsifier. This was adjusted to 1000 ml with water for injection, and then sterilized at 121 ° C. for 10 minutes to produce a phospholipid suspension. The composition of the suspension is
It contains 1.2 g of egg yolk lecithin (yolk phospholipid) and 2.25 g of glycerin, and has a calorific value of 19.8 kcal / 1.
It was 00 ml. The average particle size of the suspension is 149 nm
Met.

Suspension Example 2 Egg yolk lecithins 6, 12, and 24 g and soybean lecithins 3 and 8 g were mixed as phospholipids with 180 ml of a 5% aqueous glucose solution at about 70 ° C., respectively.
After preliminary suspension for 0 minutes, the suspension was homogenized with an ultrasonic emulsifier under ice-cooling under a nitrogen stream to obtain a suspension having a uniform particle diameter.
This was adjusted to 200 ml with water for injection, then sterilized by autoclaving, and as yolk lecithin, 3.0, 6.0 and 12.
Phospholipid suspensions of 0%, 1.5 and 4.0% soy lecithin were prepared.

Example 1 Male Wistar rats, 10 weeks old, were fasted for 3 days, and a central venous catheter was placed in the jugular vein according to a conventional method.
An N-executable animal model was created. The phospholipid suspension prepared in Suspension Example 1 was administered to four rats to confirm the efficacy. 33 ml / k of the suspension of Suspension Example 1 on day 1
g, 66 ml / kg on the second day, 100 ml / kg on the third day,
The administration was performed at 100 ml / kg on the fourth day and 100 ml / kg on the fifth day. 50% glucose preparation, 1
A nutritional supplement of TPN was prepared using a 2% amino acid formulation, an electrolyte and a multivitamin formulation and was supplemented for 5 days. A lipopolysaccharide (LPS, endotoxin) derived from E. coli was injected 0.8 m from the central venous catheter on day 4.
g / kg, a sepsis model was prepared, and dissected on day 5. Observation items are mortality (up to 5 days), blood biochemistry (platelet count, GOT, GPT, BUN), and bacterial colony count in liver tissue.

Comparative Example 1 The procedure of Example 1 was repeated, except that the amount of the glucose preparation was increased instead of the suspension prepared in Example 1. The amount of increase in glucose was set to have the same caloric content as the suspension of Example 1.

The results of the observation items for Example 1 and Comparative Example 1 are shown in Tables 1 and 2 below.

[Table 1]

As is clear from Table 1, the mortality of the rat was as high as 50% in Comparative Example 1, but 0% in Example 1.
Met. Further, it was confirmed that Example 1 suppressed a decrease in platelet count, an increase in GOT / GPT and an increase in BUN as compared with Comparative Example 1, and significantly prevented multi-organ failure due to sepsis. Also, from the results of the number of bacterial colonies in the liver, Example 1 was compared with Comparative Example 1 in the Bacterial Translocatio.
It was found that n was significantly suppressed.

Example 2 A 10-week-old male Wistar rat was placed with a central venous catheter in the jugular vein according to a conventional method. The phospholipid suspension of Suspension Example 1 was added to this rat at a rate of 100 ml / kg / day for 3 days.
It was administered for consecutive days. After the administration for 3 days, the cholesterol fraction lipoprotein in the serum was measured.

Comparative Example 2 A 10% soybean oil-fat emulsion was administered to rats prepared in the same manner as in Example 2 at a rate of 100 ml / kg / day for 3 consecutive days. After the administration for 3 days, the lipoproteins in the cholesterol fraction in the serum were measured.

Control Example 1 Male Wistar rats, 10 weeks of age, were allowed free access to water and the lipoproteins in the serum cholesterol fraction were measured.

Table 2 shows the measurement results of Example 2, Comparative Example 2 and Control Example 1.

[Table 2]

As is clear from Table 2, Example 2 is different from Comparative Example 2 and Control Example 1 in that β-lipoprotein (LD
L: low density lipoprotein) and the fractional value of the origin (chylomicron) were remarkably increased. But,
Lipoprotein X was not found.

Examples 3 to 6 Male Wistar rats were 10 weeks old and a central venous catheter was placed in the jugular vein according to a conventional method. 3. 3.0% yolk lecithin suspension, 6.0% yolk lecithin suspension, 1.5% soy lecithin suspension prepared in Suspension Example 2 for this rat;
The 0% soy lecithin suspensions were respectively 5.0 g / kg, 15.0 g / kg, 2.4 g / kg, as phospholipid amount.
The administration was continued at a rate of 3.2 g / kg for 20 hours, and after the administration was completed, the lipoprotein content of the cholesterol fraction in the serum was measured.

Control Example 2 Male Wistar rats were 10 weeks old with free access to water, and the lipoprotein content in the serum cholesterol fraction was measured.

Table 3 shows the measurement results of Examples 3 to 6 and Comparative Example 2.

[Table 3]

As is clear from Table 3, all Examples showed a remarkable increase in the lipoprotein value as compared to Control Example 2. However, lipoprotein X was not found.

Examples 7 to 9 Male Wistar rats were 10 weeks old and a central venous catheter was placed in the jugular vein according to a conventional method. The 3.0% yolk lecithin suspension prepared in Suspension Example 2 for this rat, 12.0%
The egg yolk lecithin suspension and the 4.0% soy lecithin suspension were respectively 3.0 g / kg and 12.0 g /
kg, at a rate of 4.0 g / kg for 24 hours. After that, lipopolysaccharide (LPS, endotoxin) derived from E. coli was added at 1.0 mg /
KOT was administered, and GOT and GPT in the serum 10 hours after the administration was measured.

Comparative Example 3 The same procedure as in Examples 7 to 9 was carried out except that the same amount of physiological saline was administered instead of the administration of the test solutions of Examples 7 to 9.

Table 4 shows the measurement results of Examples 7 to 9 and Comparative Example 3.

[Table 4]

As is clear from Table 4, all of the examples have significantly lower GOT and GPT values than Comparative Example 3.

Example 10 Male Wistar rats were aged 10 weeks, and a central venous catheter was placed in the jugular vein according to a conventional method. To this rat, lipopolysaccharide (LPS, endotoxin) was administered at 1.0 mg / kg from a central venous catheter, and endotoxin administration 3
After time, the phospholipid suspension of Suspension Example 1 was flowed at a flow rate of 10 ml /
Heparin was administered at 10 kg / hr for 10 hours, and heparin was collected before, 5 hours, 10 hours, 24 hours, and 48 hours after suspension administration to obtain plasma. For the measurement of plasma endotoxin, heparin plasma was diluted 10-fold with a surfactant-containing endotoxin sample pretreatment solution, and the Limulus reagent (E
S-II Test Wako) and measured using a turbidimetric time analysis method (Toxinometer ET-301).

Comparative Example 4 The same procedure as in Example 10 was carried out except that the same amount of physiological saline was administered instead of administering the suspension of Example 10.

As is apparent from FIG. 1, the endotoxin activity of Example 10 was significantly lower than that of Comparative Example 4 by administration of the test solution.

[0046]

INDUSTRIAL APPLICABILITY The present invention can use a phospholipid suspension for the prevention and treatment of sepsis, and has the effect of preventing the onset of sepsis and remarkably reducing the state of sepsis (such as multiple organ failure). Have. In addition, the present invention can be prepared much more easily than conventionally known preparations, and is not an abnormal lipoprotein mobilized by a large dose of a conventionally known soybean oil fat emulsion, but has a clear preventive and therapeutic effect. It is a parenteral preparation that mobilizes high lipoproteins. Furthermore, the present invention is a very excellent parenteral preparation which has less burden on the liver and kidney and has less side effects than the conventional fat emulsion.

Further, since the parenteral preparation of the present invention is stable when formulated and mixed with a nutritional composition used for nutritional supplementation, it can be administered via a nutritional administration route, and can be used as an emulsifier such as a fat emulsion. It is a very safe formulation that can also be used as a pharmaceutical. It is not practical to directly administer LDL or the like, which is a lipoprotein, because an ultracentrifuge is required for the preparation and there is a risk of virus infection derived from the blood of the raw material. However, the parenteral preparation of the present invention has no risk of virus infection, is stable to heat sterilization, can easily adjust its concentration, and can be mass-produced at low cost as a pharmaceutical.

[Brief description of the drawings]

FIG. 1 shows changes in plasma endotoxin activity in Example 10 and Comparative Example 4.

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Claims (7)

[Claims] 1. A preparation comprising phospholipids suspended therein, wherein when intravenously administered, said phospholipids mobilize lipoproteins in vivo and said lipoproteins endotoxin and / or endotoxin lipopolysaccharide A parenteral preparation for sepsis, characterized by inactivating a binding protein complex. 2. The lipoprotein recruited is lipoprotein X (lipoprotein X).
The parenteral preparation for sepsis according to claim 1, wherein the parenteral preparation is a lipoprotein other than protein-X: Lp-X). 3. The parenteral preparation for sepsis according to claim 1, wherein the phospholipid is at least one selected from egg yolk lecithin, soy lecithin, and synthetic lecithin. 4. The parenteral preparation for sepsis according to claim 3, wherein the phospholipid is egg yolk lecithin and / or soybean lecithin. 5. The parenteral preparation for sepsis according to claim 1, wherein the phospholipid concentration is 0.01 to 15%. 6. The parenteral preparation for sepsis according to claim 1, comprising at least one nutritional composition selected from amino acids, sugars, and electrolytes. 7. A method for preventing and treating sepsis, wherein the preparation according to claim 1 is administered intravenously to inactivate endotoxin and / or endotoxin-lipopolysaccharide-binding protein complex.