JPH10251161A - Medicine for reinforcing treating effect on edema - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject medicine capable of reinforcing the edema- treating effect of a diuretic medicine by simultaneously using human serum albumin prepared by genetic operations together with the diuretic medicine. SOLUTION: This medicine contains (A) human serum albumin (hereinafter rHSA) prepared by genetic operations as an active ingredient, and can reinforce the edema-treating effect of (B) a diuretic medicine. The component A is prepared by using a rHSA-producing bacterium, an animal cell, etc., especially a yeast as host, in particular, a yeast belonging to the genus Saccharomyces or Pichia through genetic operations. Thereby, it can be expected that treating effects on nephrotic syndrome, kidney diseases, etc., are reinforced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the pharmaceutical use of human serum albumin prepared by genetic engineering. More specifically, the present invention relates to a drug for enhancing an edema treatment effect by being used in combination with a diuretic.

[0002]

2. Description of the Related Art Human serum albumin (hereinafter referred to as HSA) is the most abundant protein in plasma, and has functions such as maintaining osmotic pressure in blood, and transporting nutrients and metabolites by binding them. Play. HS having such a function
A is used as a medicament for treating hypoalbuminemia, hemorrhagic shock, and the like due to albumin loss and reduced albumin synthesis.

[0003] Traditionally, HSA has been produced primarily as a product from a fraction of the collected blood. However, the method of producing HSA using blood as a raw material has drawbacks such as difficulties in supplying blood, uneconomical effects, and the possibility of contamination with undesired substances such as hepatitis virus. Is desired.

[0004] Under these circumstances, with the advent of recombinant DNA technology, techniques for mass production and purification of HSA by genetic manipulation have been established in place of blood-derived HSA.

[0005]

Accordingly, an object of the present invention is to provide a new pharmaceutical use for HSA prepared by genetic manipulation.

[0006]

Means for Solving the Problems The present inventors have conducted studies in consideration of the above circumstances, and as a result, it has been found that a combination of a diuretic and HSA prepared by genetic manipulation (hereinafter also referred to as rHSA) is used in the treatment of edema. The present invention has been found to be extremely useful.

[0007] That is, the present invention relates to an edema therapeutic effect enhancer containing human serum albumin prepared by genetic manipulation as an active ingredient for enhancing the edema therapeutic effect of a diuretic.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION

(1) rHSA (i) HSA obtained by genetic manipulation The rHSA-producing host prepared by genetic manipulation used in the present invention is not particularly limited as long as it is prepared through genetic manipulation, and is already described in the known literature. In addition to the above, even those that will be developed in the future can be used as appropriate. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) and animal cells that have been rendered rHSA-producing through genetic manipulation. In particular, it is preferable to use yeast, especially Saccharomyces (eg, Saccharomyces cerevisiae), or Pichia (eg, Pichia pastoris) as a host. In addition, an auxotrophic strain or an antibiotic-sensitive strain may be used. More preferably, Saccharomyces cerevisiae AH22 strain (a, his 4, leu 2, can
1), Pichia pastoris GTS115 strain (his 4) is used.

The method for preparing these rHSA-producing hosts, the method for producing rHSA by culturing the host, and the method for separating and collecting rHSA from the culture can be carried out by employing known and equivalent methods. it can. For example, as a method for preparing an rHSA producing host,
For example, a method using a normal HSA gene (Japanese Unexamined Patent Publication No.
No. 56684, No. 58-90515, No. 58-150
517), a method using a novel HSA gene (JP-A-62-29885, JP-A-1-98486), and a method using a synthetic signal sequence (JP-A-1-24).
0191), a method using a serum albumin signal sequence (Japanese Patent Application Laid-Open No. 2-167095), and a method of integrating a recombinant plasmid into a chromosome (Japanese Patent Application Laid-Open No. 3-7288).
No. 9), a method of fusing hosts with each other (Japanese Unexamined Patent Publication No.
3877 JP), a method where mutation in methanol containing medium, a mutant AOX method using a ₂ promoter (JP-A-6-90768, the 4-299984 JP), expression of HSA by Bacillus subtilis (JP-62 -251
No. 33), expression of HSA by yeast (JP-A-60-1985)
No. 41487, No. 63-39576, No. 63-744
No. 93) and the expression of HSA by Pichia yeast (JP-A-2-104290).

[0010] Among them, a method for causing a mutation in a methanol-containing medium is specifically performed as follows. That is, first, an appropriate host, preferably Pichia yeast, specifically, the GTS115 strain (NRRL accession number Y-15851)
Obtain a transformant HSA by a conventional method to AOX ₁ gene region under AOX ₁ promoter governing of introducing a plasmid having a transcription unit to express (JP 2-104
290). This transformant has a low ability to grow in a methanol medium. Therefore, this transformant is cultured in a medium containing methanol to cause mutation, and only a viable strain is recovered. At this time, the methanol concentration is, for example, about 0.0001 to 5%. The medium may be an artificial medium or a natural medium. Culture conditions are 1
5 to 40 ° C. and about 1 to 1000 hours are exemplified.

As a method for culturing an rHSA-producing host, in addition to the methods described in the above publications, a high-batch glucose or methanol or the like is supplied in small amounts in a fed batch culture (half-batch culture). A method of obtaining a high concentration of cells and a product by avoiding high-concentration substrate inhibition of the producing cells (JP-A-3-83595), and a method of enhancing the production of rHSA by adding a fatty acid to the medium ( JP-A-4-293495) and the like.

(Ii) Purification of rHSA Various methods have been studied for isolating and purifying rHSA produced by the culture treatment from components derived from host cells, culture components, and the like with sufficient accuracy. For example, as a conventional method, after subjecting a yeast culture solution containing rHSA to squeezing → ultrafiltration membrane treatment → heating treatment → ultrafiltration membrane treatment, a cation exchanger, hydrophobic chromatography, anion Method for subjecting the exchanger to a process such as column chromatography (Japanese Patent Application Laid-Open No. 5-310079, Biotechnol
ogy of Blood Proteins. 1993, Vol. 227, 293-298). Further, a method has also been reported in which the above conventional method is further subjected to a chelate resin treatment or a boric acid / salt treatment step (JP-A-6-56883, JP-A-6-2683).
No. 45789). Further, a stream line method using an adsorption fluidized bed technique after subjecting the yeast culture solution to heat treatment (Japanese Patent Application Laid-Open No. 8-116985) or the like can also be used.
That is, heat treatment of a culture solution containing a human serum albumin producing host obtained by genetic manipulation, contacting the heat-treated solution with adsorbent particles floating in a fluidized bed, and collecting the adsorbed fraction. It is a feature.

(Iii) Properties of highly purified HSA derived from genetic engineering The HSA of the present invention has a molecular weight of about 67000 and an isoelectric point of about 4.
9 single substances. The HSA is composed of a monomer and a trace amount of a dimer, and contains substantially no polymer or decomposition product.
Specifically, the total content of dimers, polymers and decomposition products is 2
% Or less is exemplified.

Further, it does not substantially contain contaminant components derived from the production host. Specifically, in the case of a 25% HSA solution,
The host-derived contaminant component having an antigenicity of 1 ng / ml or less is exemplified. Pyrogen is 1 EU / m in a similar case
l and below are exemplified.

The degree of coloring is based on the case of a 25% HSA solution.
0 / A280 is 0.015 or less, preferably 0.013 or less. Specifically, about 0.01 to 0.015 is exemplified. It is to be noted that, by using known purification methods in combination as appropriate, it is possible to obtain an HSA with a further reduced degree of coloring, that is, a low A350 / A280 value.

(Iv) Formulation of the formulation The obtained HSA is formulated by a known method (for example, heat sterilization at 50 to 80 ° C for 30 minutes or more, ultrafiltration, sterilization filtration, dispensing, freeze-drying, etc.). can do. As a preparation, specifically, a 5-25% solution of HSA (HSA
Liquid formulation having a pH of about 6.4 to 7.4 and an osmotic pressure ratio of about 1. The HSA preparation of the present invention preferably contains sodium caprylate and / or acetyltryptophan as a stabilizer. The amount of the stabilizer added is 0.01 to 0.2 M, preferably 0.02 to 0.2 M, respectively.
About 0.1M is exemplified. Further, the sodium content is, for example, 3.7 mg / ml or less. The addition time of the stabilizer is usually heat sterilization, ultrafiltration, sterilization filtration,
Before processing such as dispensing and freeze-drying. Thus, HSA not only has its storage stability, but also stabilizes in the formulation process.

Examples of containers for dispensing the HSA preparation include glass, polyethylene, and dealkalized soft glass (JP-A-4-210646).

(2) Diuretic The diuretic used in the present invention is not particularly limited, and any known drug may be used. For example, thiazide diuretics, loop diuretics (eg, furosemide, azosemide, mefluside, bumetanide, piretanide, ethacrynic acid, torasemide), anti-aldosterone agents (eg, triamterene, spironolactone, potassium canrenoate), decarbonation inhibitors ( Examples include acetazolamide), osmotic diuretics (eg, mannitol, isosorbide) and the like. Of these, a loop diuretic is preferred.
The dosage of the diuretic may be the same as before.

(3) Dosage Indication Indication: The drug of the present invention is intended to enhance the therapeutic effect of edema by diuretics, for example, nephrotic syndrome, especially hypoproteinemia, hypoalbuminemia, edema, hypertension. It enhances the therapeutic effect of those accompanied by lipemia and kidney disorders. Specifically, pharmacological actions of diuretics, for example, increase in urine volume, increase in urinary electrolyte excretion, urinary Na / K
It enhances pharmacological actions such as increasing the ratio, increasing serum oncotic pressure and decreasing ascites volume. The drug of the present invention is also useful for diuretic-resistant (refractory) patients.

RHSA is rHSA when the pharmacological action of the diuretic is exhibited.
Any mode may be used as long as rHSA is used in a situation where the pharmacological action of SA is exhibited, for example, in a situation where rHSA is present in the body, particularly in blood at the time of the pharmacological action of diuretic. Therefore, it may be administered as a mixed preparation with the diuretic, or as a separate preparation from the diuretic. The dose of rHSA is usually 20 to 50 for a 25% HSA solution once for an adult.
100% to 250 ml (HSA
5 to 12.5 g) can be slowly administered by intravenous injection or intravenous drip. In addition, it can be appropriately increased or decreased depending on age, symptoms, and weight.

[0021]

EXAMPLES The present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Example 1 Acquisition and culture of rHSA-producing yeast Pichia pastoris were carried out according to the method described in JP-A-5-317079. RHS from the resulting culture
The recovery and purification of A was performed according to the method described in JP-A-8-116985.

The prepared HSA was adjusted to a 25% solution,
After further adding sodium caprylate and acetyltryptophan to a final concentration of 0.02 M, 6
HSA can be used as an injection by heat sterilization at 0 ° C. for 30 minutes and sterilization filtration using a 0.22 μm filter (Millipore). Its composition is sodium chloride content of 3.7 mg / ml or less, p
H was 6.4 to 7.4, and the osmotic pressure ratio was about 1 (ratio to physiological saline).

Example 2 Properties of purified rHSA (containing composition) (1) HPLC analysis of purification step HSA was analyzed by HPLC gel filtration. Gel filtration analysis was performed under the following conditions. Column: TSK gel G3000SW (manufactured by Tosoh Corporation) Developing solution: 0.1 M KH ₂ PO ₄ /0.3 M NaCl buffer Detection: absorbance at a wavelength of 280 nm The purified HSA-containing composition had a single peak of the HSA monomer.

(2) Analysis of Yeast-Derived Components A culture supernatant of a non-HSA-producing yeast was roughly purified in the same manner as in the present method, and a rabbit was immunized. Of yeast-derived components present in the yeast. The measurement was performed by an enzyme immunoassay (EIA method). As a result of measurement using an HSA concentration adjusted to 25%, the amount of yeast-derived components was less than 1 ng per 250 mg of HSA.

(3) Molecular weight The molecular weight was measured by the aforementioned HPLC gel filtration method. The molecular weight of HSA in the composition containing purified HSA of the present invention is about 67.
000.

(4) Isoelectric point The isoelectric point was measured using a thin-layer polyacrylamide gel according to the method of Allen et al. [J. Chromatog., 146, 1 (1978)]. The isoelectric point of HSA in the purified HSA-containing composition of the present invention was about 4.9.

(5) Degree of coloring The degree of coloring is 280 nm, 350 nm, 450 nm, 500
The absorbance at nm is measured, and A350 / A280, A450 / A
280 and A500 / A280 were calculated. Purified HSA of the present invention
The coloring degree of the composition is about 0.015 at A350 / A280,
A450 / A280 is about 0.01, and A500 / A280 is about 0.2.
002.

(6) Measurement of pyrogen When measured by using Seikagaku Corporation's Endospecy,
The amount of pyrogen was less than 0.5 EU per 250 mg of HSA.

Test Example 1 A puromycin aminonucleoside (PAN) nephropathy model was prepared, and the edema-improving effect of combined administration of furosemide and rHSA was examined.

(1) Experimental procedure Preparation of nephrotic model Wistar male rats (47) were given PAN (20 mg).
/ Kg body weight / day) was administered subcutaneously for 5 days and kept in metabolic cages. 24-hour urine was measured from the 7th day to the 8th day from the start of PAN administration, and was used as an index for grouping. Also,
On the 8th day, the rats are self-lens (registered trademark, Maruishi Pharmaceutical)
After inhalation anesthesia was performed, the patient was fixed in the dorsal position, and the abdomen was punctured to confirm the presence or absence of ascites accumulation in each individual. The next day (P
After measuring the body weight on the 9th day after the start of AN administration), the nephrotic rats were divided into groups so that the body weight was almost uniform among the groups. As a control group (normal group), rats to which PAN was not administered were used.

Administration of Test Drug and Urine Collection Method The test drug was administered intravenously for three consecutive days with the grouping day as the first day. In the nephrosis rat-furosemide + rHSA combination group, furosemide 5 mg / kg body weight (the administration amount was 3
ml / mouse), bolus intravenous injection, and immediately after that, 1.0 g of rHSA / kg body weight / 2 hours (dose volume was 3
ml / animal) was continuously infused intravenously for 2 hours. In the nephrotic rat-furosemide alone administration group, furosemide was administered in the same manner as described above, and then physiological saline (3 ml / animal) was administered instead of rHSA. Nephrotic rats-untreated group received 3 ml of saline instead of furosemide and rHSA / animal followed by 3 ml of saline
1 / animal was continuously infused. The control group (normal group) had physiological saline 3 instead of furosemide and rHSA.
After bolus administration of 1 ml / animal, 3 ml / animal of saline was continuously infused. The urine was collected for 2 hours (2 hours urine) from the start to the end of the continuous infusion, and after measuring the urine volume, the following parameters were measured after centrifugation (3000 rpm, 10 minutes). The rats were sacrificed the day after the drug administration on the third day, and blood was collected after ascites was collected. For blood, each parameter was measured for serum obtained after centrifugation (3000 rpm, 10 minutes).

(2) Measurement Items and Methods The following parameters (i) to (iv) were measured as the above-mentioned parameters, and the results are shown in Table 1.

(I) Ascites The amount of ascites was measured by placing a tissue paper in the abdomen at the time of dissection to contain ascites, and measuring the weight.

(Ii) Blood Biochemical Examination Serum total cholesterol and serum oncotic pressure were measured using serum obtained at the time of dissection.

(Iii) Urinalysis Urine volume, urinary protein, urinary electrolytes (Na, K, Cl) and urinary creatinine were measured. Urine volume and urinary electrolytes (Na,
K, Cl) was measured on 2 hour urine on the first day of drug administration. Urinary protein and urinary creatinine were measured on 2-hour urine collected on the third day of drug administration. In addition, urine creatinine, urine protein, and serum total cholesterol were measured using a commercially available measurement kit [urine creatinine measurement: creatinine-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), urine protein measurement: micro TP-test Wako (Wako Pure Chemical Industries, Ltd.) ), Serum total cholesterol measurement: cholesterol CI
I Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.)].
Serum oncotic pressure is determined by Osmotat 050 (Gonot
ec) and the urinary electrolyte was Olympu
The measurement was performed using sNAKA-2.

(Iv) Creatinine clearance (Ccr) From urine volume, urine creatinine and serum creatinine, creatinine clearance in urine for 2 hours was calculated by the following equation. Ccr of 2 hour urine (ml / min / kg) = Cr in urine of 2 hour urine
(Mg / dl) x 2 hour urine volume (ml) / serum Cr (mg / dl) /
(2 × 60) (min) / body weight (kg)

[0038]

[Table 1]

(3) Statistical processing The fluctuations of each parameter associated with nephrosis were as follows: the normal group (physiological saline + physiological saline administration group) and the nephrotic rat-untreated group (physiological saline + physiological saline administration group). Group Stu
It was tested by the dent's-t method and evaluated by comparison. The significance between the nephrotic rat groups (untreated group, furosemide alone administration group, and furosemide + rHSA combination group) was tested by the Tukey-Kramer method. In each of the tests, a case where the risk factor was less than 5% was determined to be significant.

Test Example 2 An experiment was conducted in the same manner as in Test Example 1 except that the dose of rHSA was changed to 0.5 g / kg body weight, and similarly good results were obtained for the edema treatment enhancing effect of the present invention.

From the above results, the nephrotic rats to which rHSA and diuretic were co-administered had a remarkable decrease in ascites volume, the amount of urine and the amount of urinary Na excretion as compared to the untreated and nephrotic rats to which only furosemide was administered. Increased and increased serum oncotic pressure indicated improved edema. In addition, a decrease in serum cholesterol level was observed, and it was shown that the use of rHSA together with a diuretic enhances the therapeutic effect of hyperlipidemia. Cretinine clearance was also increased, due to an increase in osmotic pressure, an increase in circulating plasma volume, and an increase in glomerular filtration rate due to an increase in renal blood flow. Seem.

[0042]

The drug of the present invention enhances the therapeutic effect of diuretics on edema, and enhances the pharmacological effects of diuretics such as increased urine output, increased urinary electrolyte excretion, increased urine Na / K ratio, Increase pharmacological actions such as decrease in ascites volume, for example, hypoproteinemia, hypoalbuminemia in nephrotic syndrome,
It enhances the therapeutic effects of symptoms such as edema and hyperlipidemia, and kidney disorders.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiromi Tsuchiyama 2-25-1, Shodai Otani, Hirakata City, Osaka Prefecture Inside the Midori Cross Research Laboratory, Inc. (72) Inventor Minoru Tsukada 2-25, Sumida Otani, Hirakata City, Osaka Prefecture No. 1 Inside the Green Cross Central Research Institute

Claims (1)

[Claims] 1. An edema therapeutic effect enhancer for enhancing the edema therapeutic effect of a diuretic, comprising human serum albumin prepared by genetic manipulation as an active ingredient.