JP3585514B2 - Erythropoietin enhancer for anti-anemic effect - Google Patents

Description

【００１９】
Ｃ群では試験開始時に比べてヘマトクリット値の低下があり、貧血の進行が観察されたのに対し、Ａ群ではヘマトクリット値の低下が若干抑制された。Ｅ群では試験開始時よりもヘマトクリット値が増加し、貧血が抑制された。これに対し、Ｅ１／２群では試験開始時よりもヘマトクリット値が少し低下した。ところが、本発明によるＡＥ１／２群では、試験開始時よりもヘマトクリット値が増加し、その値はＥ１／２群と比較して高値となり、Ｅ群と同程度に貧血が抑制された。Ｅ群とＡＥ１／２群との間には有意差はなかった。Ｅ群とＡＥ１／３群との間にも有意差はなかった。すなわち、球形吸着炭と併用することによりｒＨｕＥＰＯは半量投与でも、また、１／３量投与でも、ｒＨｕＥＰＯのみの投与と同程度の効果があることを示した。なお、正常ラットのヘマトクリット値は、４３〜４９％である。また、摂餌量から計算した抗貧血作用増強剤（球形吸着炭）の投与量は、約１８．８ｇ／ｋｇ（ラット体重）／週であった。
【００２０】
製剤調製例１：カプセル剤の調製
前記製造例１で得た球形吸着炭２００ｍｇをゼラチンカプセルに封入してカプセル剤を調製した。
【００２１】
製剤調製例２：スティック剤の調製
前記製造例１で得た球形吸着炭２ｇを積層フィルム（構成＝グラシン／ポリエチレン／アルミニウム箔／ポリエチレン／ポリ塩化ビニリデン；厚さ＝７４±８μｍ）製スティックに充填した後、ヒートシールしてスティック剤とした。
【００２２】
【発明の効果】
ｒＨｕＥＰＯ抗貧血剤を用いる場合に、球形炭からなる本発明の増強剤を併用することにより、ｒＨｕＥＰＯの抗貧血作用は著しく増強されて、ｒＨｕＥＰＯの投与量を著しく少なくすることができる。従って、ｒＨｕＥＰＯによる副作用は、著しく軽減される。[0001]
[Industrial applications]
The present invention relates to an enhancer for an anti-anemic effect of erythropoietin, particularly recombinant erythropoietin (recombinant human EPO: hereinafter sometimes referred to as rHuEPO), comprising spherical charcoal as an active ingredient.
[0002]
[Prior art]
Advances in genetic engineering have led to the use of rHuEPO formulations for the treatment of anemia. Erythropoietin is one of the hematopoietic promoting factors existing in the living body, and erythropoietin deficiency is one of the causes of anemia. rHuEPO has been identified chemically, immunologically and biologically to be identical to human erythropoietin.
rHuEPO is a glycoprotein composed of 165 amino acid residues and having a molecular weight of about 30,000, has disulfide bonds at two positions (7-161 and 29-33), and has three N-glucoside-type sugar chains and one O-glucoside-type sugar chains are bonded to asparagine at positions 24, 38 and 83 and serine at position 126, respectively. There are various types of rHuEPO depending on the difference in sugar chain structure. Examples of rHuEPO include epogin (trade name) and Espoo (trade name) which are currently commercialized (see Iwakawa Seigo, Ayumi of Medicine, Vol. 155, No. 10, 631-633, 1990).
[0003]
[Problems to be solved by the invention]
Normally, erythropoietin levels in the blood increase when anemia becomes severe. However, in patients with renal anemia, blood erythropoietin levels are often lower than those in patients with normal renal function, and hematocrit levels increase as expected due to the presence of hematopoietic suppressors. In many cases, a large amount of rHuEPO is required in order to perform this.
However, rHuEPO causes side effects such as increased blood pressure, headache, hypertensive encephalopathy, cerebral infarction and myocardial infarction due to thrombus formation, retinal vein occlusion, shunt occlusion, and decreased dialysis efficiency. It is also said that rHuEPO-induced allergy or anaphylaxis-like symptoms include blood pressure drop, joint pain, pruritus, edema, red eye, chest tightness, bleeding spots and the like. In addition, fever, rash or liver damage may be transient and mild. Hypertension due to side effects is thought to be due to increased peripheral vascular resistance due to contraction of peripheral blood vessels and increased blood viscosity.
The present inventor has conducted intensive studies on a method for suppressing the side effects caused by rHuEPO administration. As a result, by using spherical charcoal together, the dose of rHuEPO was significantly reduced while maintaining the anti-anemic effect of rHuEPO. Can be avoided or reduced. That is, it was found that spherical charcoal enhanced the anti-anemic effect of rHuEPO. The present invention is based on these findings.
[0004]
[Means for Solving the Problems]
Therefore, the present invention relates to an erythropoietin anti-anemic potentiator comprising spherical charcoal as an active ingredient. That is, the present invention relates to a pharmaceutical preparation which enhances the anti-anemic effect of erythropoietin by using spherical charcoal in combination with erythropoietin having an anti-anemic effect.
[0005]
Hereinafter, the present invention will be described in detail.
The spherical carbon used as the active ingredient of the enhancer of the present invention is not particularly limited as long as it is a spherical activated carbon that can be used for medical treatment. Conventionally, powdered activated carbon, which has been used in medicine as an antidote, tends to cause constipation as a side effect, and constipation during pathological conditions is particularly dangerous.
The spherical coal used in the present invention has a particle size range of 0.05 to 2 mm in diameter. If it is less than 0.05 mm, there is no sufficient effect for removing side effects such as constipation, and if it exceeds 2 mm, not only is it difficult to take the drug, but also the desired pharmacological effect is not rapidly exhibited. The shape of the spherical coal is one of the important factors for obtaining the effects of the present invention, and it is necessary that the shape of the spherical coal is substantially spherical.
[0006]
For the production of spherical coal, any activated carbon raw material, for example, sawdust, coal, coconut shell, petroleum or coal-based pitches, or organic synthetic polymers can be used as a raw material, and petroleum hydrocarbons can be used. It is preferable to use It is preferable to use spherical activated carbon and / or spherical adsorbed carbon as the spherical carbon.
[0007]
The spherical activated carbon that can be used in the present invention is activated carbon having a diameter of 0.05 to 2 mm. The basic method for producing spherical activated carbon is to activate the carbonized raw material. Various known methods such as water vapor activation, chemical activation, air activation or carbon dioxide activation can be used as the activation method. The spherical activated carbon can be prepared, for example, by the following three methods. First, the first method is to granulate the powder raw material into a small spherical shape with a binder such as pitch, and then heat and calcinate at 600 to 1000 ° C. in an inert atmosphere, and subsequently carbonize in a steam atmosphere. Activate at ° C. In the second method, as described in JP-B-51-76, for example, pitches are made into small spheres in a molten state, then infusibilized with oxygen, and then the same as in the first method described above. It activates carbonization under conditions. As a third method, as described in, for example, Japanese Patent Publication No. 59-10930, a pitch is formed into a string-like pitch in a molten state, crushed, put into hot water to form a sphere, and then spheroidized. After being made infusible with oxygen, carbonization is activated under the same conditions as in the first method.
[0008]
The spherical adsorptive carbon that can be used in the present invention has a diameter of 0.05 to 2 mm, a pore size of a pore radius of 80 Å or less of 0.2 to 1.0 ml / g, and a total acidic group (A) of 0.30 to 0.30. Activated carbon having a total basic group (B) of 0.20 to 0.70 meq / g, and a total acidic group (A) / total basic group (B) of 0.40 to 2.5 meq / g. It is preferable that An example of such a spherical adsorptive carbon is a spherical carbonaceous adsorbent described in Japanese Patent Publication No. 62-11611 (U.S. Pat. No. 4,681,764).
[0009]
Spherical adsorbed carbon is prepared by subjecting spherical activated carbon having a diameter of 0.05 to 2 mm and a pore radius of 80 angstrom or less to a void volume of 0.2 to 1.0 ml / g to an oxidation treatment and a reduction treatment at a high temperature. Can be. By the oxidation and reduction treatments at a high temperature, the acidic groups and basic groups of the obtained spherical adsorbed carbon are converted into total acidic groups (A) of 0.30 to 1.20 meq / g and total basic groups (B) of 0.20 It is preferable to adjust the ratio to 0.70 meq / g and the total acidic group (A) / the total basic group (B) to the range of 0.40 to 2.5. Here, the total acidic group (A) and the total basic group (B) are physical properties quantified as follows by a conventional method.
(A) All acidic groups (A)
1 g of spherical adsorbed carbon ground to 200 mesh or less was added to 50 ml of 0.05 N NaOH solution, and after shaking for 48 hours, the spherical adsorbed carbon was filtered off and the consumption of NaOH determined by neutralization titration.
(B) All basic groups (B)
1 g of spherical adsorptive carbon ground to 200 mesh or less was added to 50 ml of 0.05N HCl solution, shaken for 24 hours, the spherical adsorptive carbon was filtered off, and the consumption of HCl determined by neutralization titration.
[0010]
The high-temperature oxidation treatment refers to high-temperature heat treatment in an oxidizing atmosphere. Pure oxygen, nitric oxide, air, or the like can be used as the oxygen source. The high-temperature reduction treatment is to perform a high-temperature heat treatment in an atmosphere inert to carbon. As the atmosphere inert to carbon, nitrogen, argon, helium, or the like, or a mixed system thereof can be used. The oxidizing heat treatment is preferably performed in an atmosphere having an oxygen content of 0.5 to 25% by volume, more preferably an oxygen content of 3 to 10% by volume, and preferably at a temperature of 300 to 700 ° C, more preferably 400 to 600 ° C. Done. The reduction treatment is preferably performed in a nitrogen atmosphere at a temperature of 700 to 1100C, more preferably 800 to 1000C.
[0011]
The enhancer of the present invention is used in combination with any erythropoietin having an antianemic effect, particularly rHuEPO. As rHuEPO, for example, various rHuEPOs produced from various mammalian cell hosts transformed with a vector containing a gene encoding at least the active site of human erythropoietin using genetic engineering techniques can be used.
[0012]
The present inventor examined the combined effect of oral administration of spherical adsorbed carbon and subcutaneous administration of rHuEPO in comparison with the case where only an effective amount of rHuEPO was subcutaneously administered to anemic rats. In the case of the combined use with, even when the rHuEPO dose was reduced to half, it was found that the same effect as when only the effective amount of rHuEPO was administered subcutaneously. That is, it was found that spherical charcoal enhanced the anti-anemic effect of rHuEPO, and was useful as an enhancer of rHuEPO anti-anemic effect. In addition, when the enhancer of the present invention was administered to normal rats, no abnormality was caused.
[0013]
The anti-anemic effect enhancer of the present invention is administered orally, and its dosage depends on the subject (animal or human), age, individual differences, medical condition, and the like. For example, the oral dose for humans is generally 0.2 to 20 g of spherical charcoal per day, and may be taken once or in 2 to 4 divided doses. The dosage may be appropriately increased or decreased depending on the condition. The spherical charcoal preparation can be provided in any dosage form such as granules, tablets, dragees, capsules, sticks, divided packages, or suspensions. When taken as a capsule, ordinary gelatin capsules or, if necessary, enteric capsules can also be used. When used as granules, tablets or sugar-coated tablets, it is necessary to release the drug into the original microparticles in the body.
[0014]
Erythropoietin may be administered by a usual method, and is generally administered as an injection intravenously, subcutaneously or intraperitoneally. An example of the administration method when only normal rHuEPO is administered is as follows.
That is, an adult is intravenously administered 3000 units at a time, three times a week, as slowly as possible. Then, when an anemia improving effect is recognized, the adult is administered twice a week, 1500 units at a time, twice or three times a week, or 3000 units at a time, as a maintenance amount. The target value for anemia improvement is around 30% in hematocrit. It is important that the hematocrit does not rise by more than 1% within one week to prevent side effects.
[0015]
The dose of erythropoietin (rHuEPO) when the enhancer of the present invention is used in combination is 5 to 90%, preferably 30 to 90% of the above dose when rHuEPO alone is used as an anti-anemic agent. Can be.
The administration time of the spherical charcoal enhancer of the present invention and the administration time of the erythropoietin antianemic agent can be freely selected and combined. For example, they may be in front of each other or may overlap. Further, only some of the periods may overlap. Further, such administration schedules can be appropriately combined or repeated.
[0016]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention.
Production Example 1: Preparation of spherical adsorptive carbon 300 g of pitch (hydrogen atom / carbon atom ratio = 0.55; pour point = 220 ° C.) and 100 g of naphthalene with a stirrer under a polarizing microscope without uneven distribution of anisotropic region The mixture was charged in an autoclave, dissolved and mixed at 180 ° C., 1200 g of a 0.5% aqueous solution of polyvinyl alcohol (Gohsenol GH-17) was added, and the mixture was vigorously stirred at 140 ° C. for 30 minutes. Got. After filtering out most of the water, the obtained spherical particles were put into an extractor, hexane was passed through to extract and remove naphthalene, and then air-dried. Then, using a fluidized bed, heated air was circulated at 25 ° C./Hr by flowing heated air to 300 ° C., and further kept at 300 ° C. for 2 hours for infusibility. Subsequently, the temperature was raised to 900 ° C. in steam, and kept at 900 ° C. for 2 hours to perform carbonization activation to obtain a porous spherical activated carbon. The diameter of the obtained spherical activated carbon was 0.05 to 1.0 mm, and the amount of voids having a pore radius of 80 Å or less was 0.755 ml / g (by a methanol adsorption method using an automatic adsorption amount measuring device). .
The carbonaceous spheres thus obtained are treated in a fluidized bed at 600 ° C. in an atmosphere with an oxygen concentration of 3% for 3 hours, then heated to 950 ° C. in a nitrogen atmosphere and kept at 950 ° C. for 30 minutes. As a result, spherical adsorbed carbon (hereinafter, referred to as sample 1) was obtained. The spherical adsorbed carbon has a diameter of 0.05 to 1 mm, a pore size with a pore radius of 80 Å or less is 0.751 ml / g (by a methanol adsorption method using an automatic adsorption amount measuring device), and all acidic groups (A ) Was 0.542 meq / g, the total basic group (B) was 0.525 meq / g, and the total acidic group (A) / the total basic group (B) was 1.03.
In an acute toxicity test by oral administration to a rat (Cpb: WU: Wistar random), no abnormality was observed even in the maximum dose (male / male rat 5000 mg / kg) according to the toxicity test method guideline (Yakuzai No. 118). Was.
[0017]
Example 1: Anti-anemic action enhancing action on anemia rat The sample 1 obtained in the above-mentioned Production Example 1 was used as spherical adsorbed carbon which is an active ingredient of the anti-anemic action enhancing agent.
As rHuEPO, epoetin alfa (genetical recombination) [Espo (trade name) of Kirin Brewery Co., Ltd.] was used. Epoetin alfa, by expression of erythropoietin genomic DNA derived from human hepatocytes, 165 amino acid residues produced in Chinese hamster ovary cells _{(C 809 H 1301 N 229 O} 240 S 5; molecular weight: 18,235.96 ) Is a glycoprotein (molecular weight; about 30,000).
Using male Wistar rats (weight around 260 g), renal anemia was developed by partial ligation of renal artery branches. On the 9th day after the operation, hematocrit values were measured and selected so that there was no bias between the groups. The control group (group C), the spherical adsorbed carbon administration group (group A), and the rHuEPO alone administration group (group E) RHuEPO half dose group (E1 / 2 group), spherical adsorbed carbon and rHuEPO half dose combined group (AE1 / 2 group), and spherical adsorbed carbon and rHuEPO1 / 3 dose combined administration group (AE1 / 3 group) In 6 groups.
The test period was 3 weeks. Each group had free access to rat chow. The drug administration for each group was specifically performed as follows.
Group C: 0.4 ml / kg of physiological saline (rat body weight) was subcutaneously administered twice a week.
Group A: Rat feed was mixed with spherical adsorbed carbon at 5% and allowed to freely ingest.
Group E: rHuEPO physiological saline solution [60 unit / kg (rat body weight)] was subcutaneously administered twice a week.
Group E1 / 2: rHuEPO physiological saline solution [30 units / kg (rat weight)] was subcutaneously administered twice a week.
AE1 / 2 group: Rat feed was mixed with 5% of spherical adsorbed charcoal and allowed to freely ingest, and rHuEPO physiological saline solution [30 unit / kg (rat body weight)] was subcutaneously administered twice a week.
AE1 / 3 group: Rat feed was mixed with 5% of spherical adsorbed charcoal and allowed to freely ingest, and rHuEPO physiological saline solution [20 unit / kg (rat body weight)] was subcutaneously administered twice a week.
After the 3-week test period, blood was collected from the jugular vein to measure the hematocrit value. Table 1 shows the results.
[0018]
[Table 1]

[0019]
In group C, the hematocrit value was lower than at the start of the test, and the progression of anemia was observed, whereas in group A, the decrease in hematocrit value was slightly suppressed. In group E, the hematocrit value was higher than at the start of the test, and anemia was suppressed. In contrast, in the E1 / 2 group, the hematocrit value was slightly lower than at the start of the test. However, in the AE1 / 2 group according to the present invention, the hematocrit value was higher than at the start of the test, the value was higher than that in the E1 / 2 group, and anemia was suppressed to the same degree as in the E group. There was no significant difference between the E group and the AE1 / 2 group. There was no significant difference between the E group and the AE1 / 3 group. That is, rHuEPO was shown to be as effective as administration of rHuEPO alone, even when administered in a half dose or in a 1/3 dose when used in combination with spherical adsorbed carbon. The hematocrit value of a normal rat is 43 to 49%. The dose of the anti-anemic effect enhancer (spherical adsorbed carbon) calculated from the amount of food consumed was about 18.8 g / kg (rat body weight) / week.
[0020]
Formulation preparation example 1: Preparation of capsule preparation A capsule preparation was prepared by enclosing 200 mg of the spherical adsorbed carbon obtained in Production Example 1 above in a gelatin capsule.
[0021]
Formulation preparation example 2: Preparation of stick preparation 2 g of the spherical adsorbed carbon obtained in the above preparation example 1 was laminated with a laminated film (structure = glassine / polyethylene / aluminum foil / polyethylene / polyvinylidene chloride; thickness = 74 ± 8 μm) After filling in a stick made from the product, it was heat-sealed to obtain a stick.
[0022]
【The invention's effect】
When the rHuEPO anti-anemic agent is used, the anti-anemic effect of rHuEPO is remarkably enhanced by using the enhancer of the present invention consisting of spherical charcoal, and the dose of rHuEPO can be significantly reduced. Therefore, side effects due to rHuEPO are significantly reduced.

Claims (3)

An erythropoietin anti-anemic potentiator comprising spherical charcoal as an active ingredient. The enhancer according to claim 1, wherein the spherical carbon is a spherical activated carbon. The enhancer according to claim 1, wherein the spherical coal is a spherical adsorption carbon.