JP2001226259A - Method for preparing a pharmaceutical preparation with oxidative deterioration prevented, method for preparing a food with oxidative deterioration prevented - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop an effective drug delivery system for preventing the oxidation of easily oxidizable drugs or nutritive substances. SOLUTION: A method for preparing a pharmacological preparation where the objective drug is prevented from oxedative deterioration is imparted by encapsulating the objective drug in lipid vesicles and by combining it with bilirubin; besides, a method for preparing food where the objective nutritive substance is prevented from oxidative deterioration is imparted by encapsulating the objective nutritive substance in lipid vesicles and by combining it with bilirubin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for preparing a pharmaceutical preparation in which bilirubin is bound to a lipid vesicle encapsulating a target drug, thereby preventing the target drug from oxidative deterioration.
The present invention also relates to a method for preparing a food, in which bilirubin is bound to a lipid vesicle enclosing a target nutrient substance to prevent oxidative deterioration of the target nutrient substance. Further, the present invention provides a pharmaceutical preparation in which bilirubin is dissolved in lipid microspheres formed by suspending oil droplets and the above-mentioned target drug or nutrient substance to prevent oxidative deterioration of the target drug or nutrient substance. A method for preparing a pharmaceutical formulation or a food.

[0002]

BACKGROUND OF THE INVENTION For many drugs or nutrients, oxidative degradation is a major problem. This is particularly acute for drugs or nutrients where oxidation is critical in nature. In preparing a drug or nutrient that is susceptible to degradation due to oxidation, it is necessary to prevent degradation by employing an appropriate drug delivery system. Oxygen carriers such as artificial blood and nucleic acid drugs have the property of undergoing oxidative degradation very quickly in vivo. Liposomes are one of the drug delivery systems used for such purposes, and are widely used as oxygen carriers such as artificial blood and carriers for nucleic acid drugs. As means for preventing such substances from being oxidatively degraded, means for encapsulating an antioxidant enzyme such as superoxide dismutase or catalase in a liposome is used.

[0003]

However, the method using an antioxidant enzyme has a problem of having antigenicity due to the use of a high-molecular-weight protein, and also has a unique oxygen species that can be eliminated. Inability to accommodate the diversity of oxygen species involved in drug oxidation. In addition, the cost is high, so that it is not an effective means. Therefore, there has been a demand for a method for suppressing the oxidation of drugs and nutrients, which has no problem of antigenicity, is inexpensive, and easy to prepare.

[0004]

Accordingly, the present inventors have focused on bilirubin, which is an antioxidant produced in vivo and has a low cost. By adsorbing a small amount of bilirubin on the membrane, it is possible to suppress the local oxidation reaction, and since it is a low molecular substance, there is no problem of antigenicity. By mixing and stirring bilirubin and lipid vesicles such as liposomes, it is possible to easily prepare bilirubin-bound liposomes. By encapsulating hemoglobin in the bilirubin-binding liposome thus prepared, the oxidation of hemoglobin by nitric oxide (NO), which is overproduced in vivo when an endotoxin is administered, was suppressed. That is, Fe ²⁺ in the hemoglobin molecule is changed to Fe ³⁺
Inhibition of the production of methemoglobin (hereinafter, referred to as methemoglobin), which is a substance converted into methemoglobin, was observed. Methemoglobin is degraded hemoglobin and has no oxygen carrying capacity due to the oxidation of iron.

[0005] The antioxidant action of bilirubin has been known for a long time, and it has been reported that its unbound form has a high affinity for lipid bilayer membranes due to its high lipophilicity (Stocker).
R, Glazer AN, Ames BN: Antioxidant activity of alb
umin-bound bilirubin.Proc.Natl.Acad.Sci.USA; 84:
5918-5922). However, there is no report showing that the substance in the liposome prevents oxidative degradation in vivo by actually binding bilirubin to the membrane.

[0006] Bilirubin has the advantage of being very inexpensive and easy to obtain, as compared to the aforementioned antioxidant enzymes. In addition, antioxidant enzymes have the ability to scavenge only certain types of active oxygen, whereas bilirubin has many types of active oxygen, such as superoxide, hydroxyl radical, lipid hydroperoxide radical, nitric oxide radical, and singlet oxygen. It has the advantage of being effective on seeds.

The antioxidant effect using bilirubin of the present invention is applied not only to a drug delivery system using vesicles such as liposomes, but also to a suspension drug delivery system such as lipid microspheres. be able to.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a method for preventing oxidative deterioration by treating lipid vesicles containing a substance susceptible to oxidative deterioration with bilirubin. That is,
Preparation of a pharmaceutical preparation comprising the steps of preparing a drug-encapsulated lipid vesicle by encapsulating a target drug in a lipid vesicle, binding bilirubin to the drug-encapsulated lipid vesicle, and preparing a bilirubin-bound drug-encapsulated lipid vesicle How to The method of the present invention makes it possible to prevent oxidative deterioration of a target drug.

In the present invention, the target drug includes hemoglobin or artificial blood which is an artificial heme complex, a nucleic acid drug or prostaglandin. In the present invention, a liposome or a lipid microcapsule can be used as the lipid vesicle. The liposomes and lipid microcapsules used here are obtained by a method generally used in the art.
Preparations can be made.

In the present invention, it is also possible to use a suspension system such as lipid microspheres instead of lipid vesicles. The present invention provides a method for preparing a drug-suspended lipid microsphere by suspending an oil droplet and a target drug, dissolving bilirubin in the drug-suspended lipid microsphere, and preparing a bilirubin-added drug-suspended lipid microsphere. A method for preparing a pharmaceutical formulation.
By this method, it is possible to prevent oxidative deterioration of the target drug. The lipid microsphere used here is obtained by a method generally used in this technical field.
Preparations can be made.

[0011] The method of the present invention can be applied to foods that are used only for drugs. That is, this is a method for preparing a food, comprising preparing bilirubin-bound lipid vesicles by binding bilirubin to lipid vesicles, and enclosing the target nutrient substance in the bilirubin-bound lipid vesicles. By this method, it is possible to prevent oxidative deterioration of the target nutrient.

In the present invention, the target nutrient includes vitamins which are retinoids or carotene. Further, in the present invention, as the lipid vesicle,
Liposomes and lipid microcapsules can be used. As described above, it is also possible to use a suspension system such as lipid microspheres instead of lipid vesicles.

[0013] Pharmaceutical preparations and foods prepared by the method of the present invention are also within the scope of the present invention. The following examples illustrate the invention, but do not limit the scope of the invention.

[0014]

EXAMPLES (Preparation of Sepsis Model) In order to examine in vivo the formation of hemoglobin by NO, a model of sepsis in rats, which seems to generate NO in large quantities, was prepared. That is, endotoxin (lipopolysaccharide: LPS, Escherichia coli endotoxin O111B4: manufactured by Sigma) is dissolved in physiological saline to a final concentration of 2 mg / ml, and a dose of 4 mg / kg is administered to rats by intraperitoneal injection. did.

(Ex vivo perfusion of liver) N by LPS treatment
To study the O of occurrence, NO ₂ of perfusate was perfused livers ^- was measured. Since NO is a radical that decomposes rapidly and cannot be measured, the amount of NO ₂ ⁻ , a metabolite of NO that is relatively stable, was measured. That is, the liver of the rats to which the control and LPS were administered was excised, and the liver was perfused in vitro with a Krebs Ringer solution containing 30 μM sodium taurocholate,
NO of perfusate ₂ ^- it was determined. The perfusate passed through the portal vein to the liver.

[0016] (NO ₂ ^- measurement) were collected venous perfusate obtained from perfused liver as described above, NO ₂ ^- and for reacting a 2,3-diaminonaphthalene, NO by fluorescence ₂ ^-
Was quantified. The reaction produced 1- (H) -naphthotriazole as a fluorescent substance. The fluorescence at 450 nm at an excitation light of 365 nm was measured by a fluorescence spectrophotometer according to a method already reported (Misko TP, Schilling RJ, Salvemini D,
Moore WN, Currie MG: A fluorometric assay for the
measurement of nitrite in biological samples.Anal
yt Biochem1993; 21: 11-18.). At this time, the calibration was performed using a dilution series of NaNO _{2 having} a known concentration.

In this study, a system using aminoguanidine having an action of suppressing inducible NO synthase (iNOS) was prepared and compared. The disappearance of the fluorescence in the presence of aminoguanidine indicates that the NO ₂ ⁻
Indicates that it is derived from NO. The control or LPS-treated liver was perfused extracorporeally and perfused for 15 minutes until the oxygen consumption of the liver was stabilized, and aminoguanidine was added to the perfusate at a final concentration of 5 mM.
In this case, the venous perfusate was collected 20 minutes after the administration of aminoguanidine and used for NO ₂ ⁻ measurement.

N in the liver perfusate 6 hours after LPS treatment
O ₂ ^- the results of measurement of, shown in Figure 1, was compared to a control without administration of LPS. In FIG. 1, the column on the left (black) after 6 hours of LPS treatment with the control shows the results without aminoguanidine, and the column on the right (white).
Column shows the results in the presence of aminoguanidine. FIG.
As a result, NO ₂ ⁻ was not detected in the control liver perfusate, but significant production of NO ₂ ⁻ was observed in the liver perfusate of the group 6 hours after LPS treatment. In the group of 24 hours after LPS treatment, NO ₂ ^- production of was slightly reduced. LPS treatment NO ₂ by ^- product of, as seen in the right column, was inhibited in the presence of aminoguanidine to control levels. Therefore, it was confirmed that NO ₂ ⁻ derived from iNOS was measured.
No difference was observed in the group (6 hours after LPS treatment) from which the liver Kupffer cells indicated by KC (-) were removed.

(Induction of NO Synthase) In order to further support the production of NO, the induction of iNOS during LPS treatment was examined. After LPS administration, the liver tissue frozen with liquid nitrogen was crushed every hour, and treated with a homogenizer. Furthermore, after denaturing the protein with mercaptoethanol, Western blotting was performed, and the result of antigen detection using a commercially available (Amersham) antibody against iNOS is shown in FIG.

As can be seen in the third lane in FIG. 2, iNOS was significantly induced 6 hours after LPS administration. And as seen in the fifth lane, iNOS expression was maintained even after 24 hours. The fourth lane is the result of the sample from which the Kupffer cells of the liver were removed, but no difference was observed in the expression level of iNOS. Considering the results of FIG. 1 and FIG. 2 together, it was strongly shown that NO was generated by LPS administration. Therefore,
The formation of HbV by the NO thus generated was examined.

As mentioned above, NO promotes the production of methemoglobin, a product of oxidative degradation of hemoglobin. The effect of treating the hemoglobin endoplasmic reticulum (HbV) encapsulated with liposomes with bilirubin on the metformation of hemoglobin contained in HbV by NO was examined. HbV is a liposome having an average diameter of about 250 nm and was prepared based on a previously reported method. The method for preparing HbV is described in the literature (Sakai H, Hamada K, Takeoka S,
Nishide H, Tsuchida E: Physical properties ofhemog
lobin vesicles as red cell substitutes.Biotechno
l.prog. 1996; 12: 119-125).

(Preparation method of bilirubin-introduced HbV) Since bilirubin is hardly soluble in water, it is dissolved in 1N NaOH, then diluted and adjusted for pH to obtain a bilirubin solution (1 mM, pH 7.4 physiological saline). And This bilirubin solution was added to an HbV dispersion ([Hb] = 10 g / d).
L) to 5 μM. Furthermore, 10
After stirring at 12 ° C. for 12 hours, the mixture was washed by ultracentrifugation to obtain bilirubin-bound HbV.

(Effect of bilirubin on methemoglobin formation by sepsis model) HbV was administered 6 hours after LPS administration to rats in a septic shock model administered LPS. HbV was administered intravenously in an amount equivalent to 10% of the blood volume in hemoglobin equivalent. HbV was prepared as a solution having a hemoglobin concentration of 10 g / dl, and an amount corresponding to 0.6% of the body weight of the rat (1.8 ml for a 300 g rat).
Was administered over 2 minutes. Immediately after the end of the administration, 0.4-0.6 ml of blood was collected from the carotid artery every 2 hours, immediately centrifuged to collect the supernatant containing HbV, and the ratio of methemoglobin to the total amount of hemoglobin in HbV (% ) Was measured spectrophotometrically.

That is, the methemoglobin conversion rate in HbV was calculated from an ultraviolet / visible absorption spectrum. Since it is difficult to destroy the parcel with a surfactant, and the general cyanomethemoglobin method causes an error in the measured value,
The measurement was performed by a non-destructive method. First, the collected blood was placed in a hematocrit tube and centrifuged to obtain a suspension of HbV in the supernatant. Approximately 20 μl was collected, diluted in 4 ml of physiological saline, and placed in a cuvette that could be sealed with a rubber tube having a length exceeding 1 cm. When deoxygenated by aeration with nitrogen for 5 minutes, a two-component system consisting of deoxyhemoglobin and methemoglobin is obtained.
And 405 nm, the methation ratio was calculated from the absorbance ratio. HbV with 100% deoxyhemoglobin and HbV with 100% deoxyhemoglobin
Was prepared, the spectrum was measured while changing the mixing ratio, and a calibration curve was prepared. The method of preparing the calibration curve is described in detail in the literature (Hamada et al., Artificial Blood vol. 3, 96-101, 199).
Five).

FIG. 3 shows the measurement results of the methation ratio. 3. In FIG. 3, .largecircle. And .circle-solid. Are the results of rats to which LPS was not administered, and .largecircle. Indicates the methionization rate of HbV not binding bilirubin, and .circle-solid. □ and ■ indicate the results of rats to which LPS was administered, □ indicates the methionization rate of bilirubin-bound HbV, and ■ indicates the bilirubin-conjugated HbV methation rate. The horizontal axis shows the time since HbV was administered.

HbV administered in the present sepsis model was 2
Almost 25% in time and about 70% in 4 hours. In other words, it was clarified that even if this drug was administered to the pathological condition of shock, it was rapidly converted to methemoglobin having no oxygen carrying ability (□). On the other hand, H with bilirubin bound to the membrane
In bV, the methation rate was significantly suppressed between 2 and 4 hours (■). On the other hand, when HbV was administered to normal rats, the methation in 6 hours was about 30%, and even if bilirubin was bound, no significant inhibitory effect on the methation rate was observed. This suggests that NO specifically increased by endotoxin shock plays an important role in hemoglobin metformation, and that bilirubin can suppress the promotion of metformation by this.

[0027]

Industrial Applicability According to the present invention, there is provided a method for preparing a pharmaceutical preparation in which a target drug is encapsulated in a lipid vesicle to which bilirubin is bound, and the oxidative deterioration of the target drug is prevented. Further, the present invention provides a method for preparing a food, in which a target nutrient substance is encapsulated in a lipid vesicle to which bilirubin is bound, and oxidative deterioration of the target nutrient substance is prevented.

[Brief description of the drawings]

FIG. 1 is a graph showing production of NO ₂ ⁻ by administration of LPS.

FIG. 2 is a photograph of Western blotting showing induction of iNOS by administration of LPS.

FIG. 3 is a graph comparing the change over time of the HbV met-formation rate with LPS administration with and without bilirubin treatment.

Continued on the front page F term (reference) 4B018 MD07 MD24 ME06 4C076 AA61 CC14 CC23 CC29 CC40 CC41 CC50 DD63 FF51

Claims (16)

[Claims] The method comprises the steps of: preparing a drug-encapsulated lipid vesicle by encapsulating a target drug in a lipid vesicle; and binding bilirubin to the drug-encapsulated lipid vesicle to prepare a bilirubin-bound drug-encapsulated lipid vesicle. A method for preparing a pharmaceutical preparation, which comprises preventing oxidative deterioration of the target drug. 2. The method for preparing a pharmaceutical preparation according to claim 1, wherein the lipid vesicle is a liposome or a lipid microcapsule. 3. The method for preparing a pharmaceutical preparation according to claim 1, wherein the target drug is hemoglobin or artificial blood which is an artificial heme complex, a nucleic acid drug or prostaglandin. 4. Prepared by the method of claim 1,
Pharmaceutical preparations. 5. A pharmaceutical preparation comprising a lipid vesicle, a drug of interest encapsulated inside the lipid vesicle, and bilirubin bound to the outside of the lipid vesicle. 6. A drug suspension lipid microsphere is prepared by suspending an oil droplet and a target drug, and bilirubin is dissolved in the drug suspension lipid microsphere to prepare a drug suspension lipid microsphere with bilirubin added. A method for preparing a pharmaceutical preparation, comprising the steps of preventing oxidative deterioration of the target drug. 7. The method for preparing a pharmaceutical preparation according to claim 6, wherein the target drug is hemoglobin or artificial blood which is an artificial heme complex, a nucleic acid drug or prostaglandin. 8. Prepared by the method of claim 6, wherein
Pharmaceutical preparations. 9. A nutrient substance-encapsulated lipid vesicle is prepared by enclosing a target nutrient substance in a lipid vesicle, and bilirubin is bound to the nutrient substance-encapsulated lipid vesicle to prepare a bilirubin-bound nutrient substance-encapsulated lipid vesicle. A method of preparing a food, comprising preventing the oxidative deterioration of the target nutrient substance. 10. The method of preparing a food according to claim 9, wherein the lipid vesicle is a liposome or a lipid microcapsule. 11. The method for preparing a food according to claim 9, wherein the target nutrient is a vitamin such as retinoid or carotene. 12. A food product prepared by the method of claim 9. 13. A food comprising a lipid vesicle, a target nutrient substance encapsulated in the lipid vesicle, and bilirubin bound to the outside of the lipid vesicle. 14. A nutrient suspension lipid microsphere prepared by suspending an oil droplet and a target nutrient, and dissolving bilirubin in the nutrient suspension lipid microsphere. A method for preparing a food, comprising preventing oxidative deterioration of the target nutrient substance. 15. The method for preparing a food according to claim 14, wherein the target nutrient is a vitamin such as retinoid or carotene. 16. A food product prepared by the method of claim 14.