WO2021065768A1

WO2021065768A1 - Space-forming agent, subcutaneous injection preparation, and space-forming method

Info

Publication number: WO2021065768A1
Application number: PCT/JP2020/036537
Authority: WO
Inventors: まゆ秦; 恵子大津
Original assignee: テルモ株式会社
Priority date: 2019-09-30
Filing date: 2020-09-28
Publication date: 2021-04-08
Also published as: JPWO2021065768A1

Abstract

Provided is a preparation which is co-administered with a protein preparation and can increase the administration volume of the protein preparation even when administered at a low concentration. This space-forming agent forms, under the skin, a space for the subcutaneous administration of a protein preparation including chondroitinase.

Description

Space forming agent, subcutaneous administration preparation and space forming method

The present invention relates to a space-forming agent, a subcutaneously administered preparation, and a space-forming method.

With the development of gene recombination technology, various active proteins have come to be provided as preparations. In particular, with the progress of antibody engineering, chimeric antibodies and humanized antibodies have been put into practical use, and have achieved great therapeutic effects as antibody drugs. Conventionally, protein preparations typified by such antibody drugs have been generally administered by intravenous injection, but in recent years, in consideration of patient convenience, preparations provided as subcutaneous administration that can be self-administered have been used. It has increased.

Such subcutaneous administration reduces the bioavailability of the drug (the rate at which it reaches the systemic circulation) compared to intravenous injection. In addition, since extracellular matrix such as hyaluronic acid and chondroitin sulfate is present in the subcutaneous tissue, the drug is more difficult to transfer into the blood, especially when a high molecular weight drug such as an antibody drug is subcutaneously administered.

On the other hand, since less than 2 mL of protein preparation can usually be administered subcutaneously, it is extremely difficult to administer a high-dose protein preparation in a single dose. Therefore, there is a problem that frequent administration of the protein preparation is required and the convenience of the patient is lowered. As one of the methods for solving this problem, it has been attempted to reduce the administration volume by increasing the concentration of the protein to administer the protein preparation.

However, in such a high-concentration protein preparation, the chances of exposure to the gas-liquid interface and interaction between protein molecules increase, and there is a high possibility that aggregates will be generated. The generation of protein aggregates may cause the formulation to become highly viscous, reducing the permeability of the needle and increasing the patient's pain during administration.

Against this background, Patent Document 1 shows that subcutaneous administration of soluble hyaluronidase glycoprotein facilitates administration of a high-concentration drug to the subcutaneous tissue. Patent Document 1 states that the hyaluronidase rapidly depolymerizes hyaluronan HA in the extracellular space, thereby increasing the volume of the drug administered into the subcutaneous tissue.

U.S. Pat. No. 9,345,661

As described in Patent Document 1, by increasing the volume (space) in which the drug is administered into the subcutaneous tissue, the dose of the drug can be increased and the concentration of the drug can be reduced to some extent.

However, Patent Document 1 uses an enzyme having a high concentration of soluble hyaluronidase glycoprotein of 2000 U / mL. This suggests that it is necessary to use soluble hyaluronidase glycoprotein at a certain high concentration in order to exert the effect of increasing the space volume in the subcutaneous tissue. When such a high concentration of enzyme is used, there is a concern that the enzyme itself may cause inflammatory or immunogenicity.

Therefore, an object of the present invention is to provide a preparation that is co-administered with a protein preparation and that can increase the administration volume of the protein preparation even at a low concentration. Another object of the present invention is to provide a preparation that is co-administered with a protein preparation and that can enhance the blood transferability of the protein preparation even at a low concentration. ..

The present inventors have clarified that by using chondroitinase, an effective space can be formed under the skin even at a low concentration of administration. It was also clarified that the use of chondroitinase can improve the bioavailability of protein preparations even at low concentrations. Based on these findings, the present inventors have completed the present invention.

The preferred embodiment of the present invention is as follows.

(1) A space-forming agent containing chondroitinase that forms a space subcutaneously for subcutaneous administration of a protein preparation.

(2) The space-forming agent according to (1), wherein the protein preparation has a molecular weight of 10 kDa or more.

(3) The space-forming agent according to (1) or (2), wherein the active ingredient of the protein preparation is an antibody.

(4) The space-forming agent according to any one of (1) to (3), which is administered at the same time as the protein preparation.

(5) Subcutaneous administration preparation containing 0.1 to 100 U / mL chondroitinase and protein preparation.

(6) The subcutaneously administered preparation according to (5), wherein the protein preparation has a molecular weight of 10 kDa or more.

(7) The subcutaneously administered preparation according to (5) or (6), wherein the active ingredient of the protein preparation is an antibody.

(8) The subcutaneously administered preparation according to any one of (5) to (7), wherein the chondroitinase and the protein preparation are mixed.

(9) A space forming method for subcutaneously administering a protein preparation, which comprises subcutaneously administering chondroitinase.

It is a schematic diagram which shows the administration method of a decomposition agent and IgG in an Example. It is a micrograph of the porcine skin 0 day after administration of only the IgG preparation. It is a micrograph of the porcine skin 0 day after the simultaneous administration of chondroitinase ABC and IgG preparation. It is a graph which shows the time passage of the insertion pressure at the time of simultaneous administration of a decomposing substance and IgG. It is a graph which shows the time lapse of the insertion pressure when the composition (mixture) of a decomposing substance and IgG is administered. It is a graph which shows the time lapse of the human IgG concentration in plasma after subcutaneous administration of each test substance.

Hereinafter, embodiments of the present invention will be described. The present invention is not limited to the following embodiments. In the present specification, "X to Y" indicating a range means "X or more and Y or less", and unless otherwise specified, operation and measurement of physical properties are performed at room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH. Perform under the conditions of.

<Space forming agent>
The first embodiment of the present invention is a space-forming agent containing chondroitinase, which forms a space subcutaneously for subcutaneous administration of a protein preparation.

According to the space-forming agent of the present embodiment, the subcutaneous administration restriction amount of the protein preparation can be widened even when the protein preparation is administered at a low concentration. Further, according to the space-forming agent of the present embodiment, it is possible to enhance the subcutaneous transferability of the protein preparation even at a low concentration.

The space-forming agent is used in combination with a protein preparation that is subcutaneously administered. For this reason, the space-forming agent is also usually administered subcutaneously. Preferably the space-forming agent is injected subcutaneously. The term "subcutaneous administration" means delivery by any suitable means (eg, injection) such that the therapeutic agent is delivered directly through the skin to the subcutaneous cavity. As used herein, the term "subcutaneous cavity" means connective tissue under the skin. This excludes blood vessels, blood flow and internal organs.

The pH of the space-forming agent when the space-forming agent is injected subcutaneously is not particularly limited, but can be neutral or acidic (pH is 8.0 or less), for example, 4 to 8. .. The pH can be adjusted by a method known per se, for example, using a buffer or a pH adjuster.

Subcutaneous administration of the space-forming agent can be administered to the patient via any suitable route. For example, it is administered to a patient as a bolus or by a subcutaneous route by continuous infusion over a period of time. Further, the space-forming agent may be administered as a single dose or as a frequent dose.

The space-forming agent refers to a preparation in which the insertion pressure after administration (preferably 2 minutes after administration) is lower than that of the control (protein preparation only). Preferably, the insertion pressure after administration of the space-forming agent is 95% or less of the control, more preferably 90% or less, still more preferably 85% or less. The insertion pressure after administration of the space-forming agent is not particularly limited, but may be, for example, 10% or more, and may be 20% or more. The insertion pressure can be measured according to the method described in the following Examples. Further, in the following examples, the insertion pressure after administration of the space forming agent is about 50% (or less) of the control on average.

The space-forming agent is used in combination with a protein preparation that is subcutaneously administered. At this time, any form may be used, such as a composition containing a space-forming agent and a protein preparation (single preparation), or a combination of separately formulating the space-forming agent and the protein preparation (drug kit).

The order of administration of the space-forming agent and the protein preparation is not particularly limited, and the space-forming agent and the protein preparation may be administered at the same time or may be administered at different times. When the space-forming agent is administered at different times, it is preferable to administer the protein preparation after the space-forming agent is administered. Since the formed space can be effectively used for administration of the protein preparation and the effect of improving the transferability of the protein preparation into blood can be easily obtained, it is preferable to administer the space-forming agent and the protein preparation at the same time. That is, a preferred form is a space-forming agent that is administered at the same time as the protein preparation. The space-forming agent administered at the same time as the protein preparation is a combination of the space-forming agent and the protein preparation in the form of a composition (single preparation); the space-forming agent and the protein preparation are separately formulated. , A form in which a space-forming agent and a protein preparation are co-administered;

In addition, the mass (mg / kg / Day) ratio of the doses of the space-forming agent and the protein preparation is appropriately determined according to each individual case in consideration of the patient's symptoms, age, sex, and the like. When the space-forming agent and the protein preparation are in liquid form, the administration volume ratio of the space-forming agent and the protein preparation is not particularly limited, but the space-forming agent: protein preparation = 1: 5 to 100. Is preferable.

[Chondroitinase]
The chondroitinase is not particularly limited, and may be any of chondroitinase ABC, chondroitinase AC I, chondroitinase AC II, chondroitinase B, and chondroitinase C. Of these, chondroitinases are chondroitin sulfate ABC, chondroitinase AC I, chondroitinase AC II, and chondroitinase C, because the effects of the present invention are likely to be exhibited. It is more preferable that the substrates are hyaluronic acid and chondroitin sulfate, chondroitinase ABC, chondroitinase AC I, and chondroitinase AC II, and chondroitinase ABC is particularly preferable. Further, the chondroitinase may be a recombinant type having at least 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more homology with the wild type chondroitinase. Good.

As the chondroitinase ABC, one derived from Proteus vulgaris can be used, or a commercially available product can be used.

When the space-forming agent is in a liquid state, the concentration of chondroitinase is not particularly limited as long as the chondroitinase is dissolved, but it is usually 0.1 to 100 U / mL in consideration of the dose and the like. Considering the convenience of subcutaneous administration, the space-forming agent is preferably liquid. As used herein, the term "liquid" refers to a liquid having fluidity at 25 ° C. More specifically, when the object is tilted by 45 °, the shape cannot be held for 10 minutes or more, and the shape changes.

[Protein preparation]
As used herein, a "protein preparation" contains an active ingredient consisting of a protein. Examples of the active ingredient include antibodies, blood coagulation / fibrinolytic factors, hormones, enzymes, cytokines, interferons, serum proteins, vaccines, erythropoetins, fusion proteins and the like.

The molecular weight of the protein, which is the active ingredient of the protein preparation, is preferably 10,000 Da or more, and in the preferred order, 20,000 Da or more and 50,000 Da or more. In the case of subcutaneous administration, it is necessary to increase the concentration of the protein preparation due to the restricted dose, but it is difficult to increase the concentration of a protein having a high molecular weight due to aggregation. Moreover, due to its high molecular weight, its absorbability in blood is also low. On the other hand, when the protein preparation is subcutaneously administered, the subcutaneous administration volume of the protein preparation can be increased by co-administering with the space-forming agent of the first embodiment, and it is not necessary to increase the concentration of the protein preparation. In addition, it is possible to enhance the subcutaneous transferability of the protein preparation into the blood. The upper limit of the molecular weight of the protein is not limited, but is preferably 1,000,000 Da or less. When the molecular weight of the protein is in the above range, the aggregation of the protein is effectively suppressed.

Above all, it is preferable that the active ingredient of the protein preparation is an antibody. Since the antibody has a large molecular weight, it is difficult to increase the concentration by aggregation. Moreover, due to its high molecular weight, its absorbability in blood is also low. By co-administering with the space-forming agent of the first embodiment, the limitation of the subcutaneous dose of the antibody can be widened, and the transferability of the antibody from the subcutaneous blood can be enhanced.

Examples of the antibody include antibody drugs. As used herein, the antibody drug includes not only a drug containing an artificially prepared antibody that specifically binds to a disease-related molecule, but also an immunoglobulin preparation purified from human plasma. Target molecules in antibody drugs are various and are not particularly limited, such as cell surface molecules such as PD-1, PD-L1, PD-L2, TNF-α, VEGF, and CD20. The type of antibody may be any of mouse antibody, chimeric antibody, humanized antibody and human antibody, but humanized antibody or human antibody is preferable. Examples of antibody drugs include immune checkpoint inhibitors, antibody drugs that specifically react with cancer antigens and have ADCC and ADCP activities, and specifically, anti-PD-1 antibody, anti-PD-L1 antibody, and the like. Immunocheckpoint inhibitors such as anti-CTLA4 antibody; antibody drugs having ADCC and ADCP activity by specifically reacting with cancer antigens such as anti-CD20 antibody, anti-HER2 antibody, and anti-EGFR antibody.

More specifically, antibody drugs include adalimumab, muromonab-CD3, trastuzumab, rituximab, paribismab, infliximab, basiliximab, tosirizumab, gemtuzumab ozogamicin, bevacizumab, iburizumab butyximab, cetuximab, cetuximab. , Ecrizumab, Panitumumab, Ustekinumab, Golimumab, Kanakinumab, Denosumab, Adalimumab, Cetuximab, Mogamurizumab, Celtrizumab Pegor, Ofatumumab, Pertuzumab, Trastuzumab Katsumakisomab, Adalimumab, Edrecolomab, Absiximab, Siltuximab, Dakrizumab, Efarizumab, Obinutuzumab, Bedrizumab, Pembrolizumab, Ixekizumab, Jiridabumab, Ipilimumab, Bevacizumab, Ipilimumab, Bevacizumab

The content of the protein preparation and physical characteristics such as pH are not particularly limited, and can be appropriately adjusted according to the type of protein preparation used.

In addition to the above components, protein preparations include stabilizers, solvents (eg physiological saline, buffers, water for injection, etc.), lysis aids, isotonic agents (eg, sodium chloride, glucose, glycerin, etc.) as required. ), Additives such as pH adjusters, soothing agents, reducing agents, antioxidants and the like.

As additives, sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene beeswax derivative, polyoxyethylene lanolin derivative, polyoxyethylene fatty acid amide, lecithin, glycerophospholipid, sphingolinlipid, Nonionic surfactants such as sucrose fatty acid esters, surfactants such as alkyl sulfates, polyoxyethylene alkyl ether sulfates, anionic surfactants such as alkyl sulfosuccinates; leucine, tryptophan, serine, glutamate, Amino acids such as arginine, histidine, lysine, methionine, phenylalanine, acetyltryptophan; phosphates such as sodium monohydrogen phosphate and sodium dihydrogen phosphate, citrates such as sodium citrate; polyoxyethylene sorbitan monooleate (polyoxyethylene sorbitan monooleate) Polysorbate 80) and / or polyoxyethylene sorbitan monolaurate (polysorbate 20), cremofol, ethanol, sodium dodecilibenzene sulfonate; polyethylene glycol; dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, Examples include sugars such as mannose, maltose, shoe chlor, and raffinose.

Protein preparations are usually liquid in consideration of convenience when administered subcutaneously. The concentration of the protein preparation is appropriately set in consideration of the dose and solubility of the drug.

The dose of the protein preparation can be appropriately adjusted in consideration of the type, use, usage pattern of the protein preparation; patient's symptom, age, sex, etc.

[Additive]
In addition to chondroitinase, space-forming agents include solvents (eg saline, water for injection, buffers, etc.), membrane stabilizers (eg cholesterol, etc.), isotonic agents (eg, sodium chloride, glucose, glycerin, etc.), It may contain antioxidants (eg tocopherols, ascorbic acid, glutathione, etc.), preservatives (eg chlorbutanol, parabens, etc.) and the like.

<Subcutaneous preparation>
A second embodiment of the present invention is a subcutaneously administered formulation comprising 0.1-100 U / mL chondroitinase and protein formulation. Here, the chondroitinase and the protein preparation are as described in the first embodiment. According to this embodiment, even if a low concentration of chondroitinase is administered, the subcutaneous administration restriction amount of the protein preparation can be widened. Further, according to the present embodiment, even if a low concentration of chondroitinase is administered, the transdermal transferability of the protein preparation into the blood can be enhanced.

Examples of the subcutaneously administered preparation include a composition containing a chondroitinase and a protein preparation (single preparation), a combination of separately formulating the chondroitinase and the protein preparation (drug kit), and the like. A preferred form is a composition (single formulation) containing chondroitinase and a protein formulation. By making the composition (single preparation), the space formed by chondroitinase can be effectively used for administration of the protein preparation, and the effect of improving the blood transferability of the protein preparation can be easily obtained. In the case of a combination of separately formulated chondroitinase and protein preparation (drug kit), the administration order of chondroitinase and protein preparation is not particularly limited, and chondroitinase and protein preparation may be administered at the same time. However, they may be administered at different times. In addition, when the administration is performed with a time lag, it is preferable to administer the protein preparation after the administration of chondroitinase. It is preferable that chondroitinase and a protein preparation are administered at the same time because the formed space can be effectively used and the effect of improving blood transferability can be easily obtained. Here, "the chondroitinase and the protein preparation are administered at the same time" means that the subcutaneously-administered preparation is a composition containing the chondroitinase and the protein preparation; the subcutaneously-administered preparation is a chondroitinase and a protein preparation. It is a combination of separately formulated protein preparations, and includes both a form in which chondroitinase and a protein preparation are administered at the same time.

The subcutaneously administered preparation is preferably a subcutaneous injection preparation because it can be efficiently administered subcutaneously. The subcutaneously administered preparation is preferably a subcutaneous injection liquid preparation.

The pH when the subcutaneously administered preparation is a subcutaneous injection liquid preparation is not particularly limited, but can be neutral or acidic (pH is 8.0 or less), for example, 4 to 8. The pH of the liquid preparation can be adjusted by a method known per se, for example, using a buffer or a pH adjuster.

Subcutaneous preparation means a drug that is subcutaneously administered and used for the prevention / treatment / diagnosis of any disease in mammals (humans, monkeys, rats, etc., preferably humans).

Subcutaneous administration of the preparation can be administered to the patient via any suitable route. For example, it is administered to a patient as a bolus or by a subcutaneous route by continuous infusion over a period of time. In addition, the subcutaneous administration preparation may be administered once or frequently. In home care, it is often given frequently at home. Since the subcutaneous administration preparation of the present embodiment can increase the administration volume per administration, the number of administrations can be reduced and the burden on the patient is small.

The content of chondroitinase in the subcutaneously administered preparation is 0.1 to 100 U / mL. Chondroitinase can effectively form a space under the skin even by subcutaneous administration at a low concentration of 100 U / mL or less. Therefore, the dose (volume) of the protein preparation can be increased by co-administering with the protein preparation. In addition, chondroitinase can enhance the subcutaneous transferability of protein preparations even when administered at a low concentration of 100 U / mL or less. Further, when the dose of chondroitinase is 100 U / mL or less, there is little possibility that the dispersibility of the protein is lowered due to the excessive chondroitinase that does not contribute to the space forming ability or the degradation product of chondroitinase.

Here, the content of chondroitinase in the subcutaneously administered preparation is the concentration in the subcutaneously administered preparation. In the case of a combination of separately formulated chondroitinase and protein preparation (drug kit), it is the content of chondroitinase with respect to the total amount of both. For example, in the case of a drug kit containing 1 mL of 50 U / mL chondroitinase and 9 mL of protein preparation, the content of chondroitinase is 5 U / mL. The content of chondroitinase in the subcutaneously administered preparation is preferably 0.1 to 80 U / mL, more preferably 0.5 to 50 U / mL. As described above, since the enzyme itself is a foreign substance to the living body, there is a concern that the enzyme itself may cause inflammatory or immunogenicity, and the concentration is required to be as low as possible. The present invention is based on the finding that chondroitinase can form a space under the skin at a very low concentration, and chondroitinase has a low concentration of about 1/100 as compared with, for example, hyaluronidase. Shows the same space-forming ability in (see Examples below).

The dose of the protein preparation in the subcutaneous preparation is appropriately determined according to each individual case in consideration of the patient's symptoms, age, sex, etc. At this time, when chondroitinase and a protein preparation are used in combination, the subcutaneous dose restriction amount of the protein preparation can be increased at one time. Therefore, it can be set to a higher dose (higher volume) than when it is usually administered subcutaneously alone. For example, the volume can be increased up to about 10 times the maximum dose (volume) when the liquid protein preparation is administered alone. In addition, the administration volume of the protein preparation can be 2 mL or more per administration, and can be 2 to 20 mL.

When a chondroitinase and a protein preparation are used in combination, the subcutaneous dose limit of the protein preparation can be increased, so the administration volume of the subcutaneous preparation should be 2 mL or more per administration. It can be 2 to 20 mL.

In addition, the mass (mg / kg / Day) ratio of the doses of the protein preparation and chondroitinase is also appropriately determined according to each individual case in consideration of the patient's symptoms, age, sex, and the like.

In addition, the subcutaneously administered preparation is preferably administered in a fixed cycle. As the administration cycle, it is preferable to appropriately adjust the administration cycle so as to be suitable for concomitant use. The specific administration frequency, dose, infusion administration time, administration cycle, etc. are appropriately determined according to individual cases in consideration of the patient's symptoms, age, gender, and the like. In the subcutaneous administration preparation of the present embodiment, the administration volume of a single protein preparation can be increased by co-administering chondroitinase, so that the administration interval of the subcutaneous administration preparation can be extended, which imposes a burden on the patient. It will be dramatically reduced. As an administration schedule, for example, in the case of a single protein preparation, the administration interval can be extended from 100 mg (1 mL) per week to 400 mg (4 mL) per month in the case of a subcutaneous administration preparation.

Another aspect of the present invention is a method for treating or preventing a disease, which comprises administering an effective amount of chondroitinase and an effective amount of a protein preparation to a subject in need of treatment or prevention. In particular, it is preferable that the disease is cancer.

The above-mentioned subjects are preferably mammals, and particularly preferably humans.

<Subcutaneous space formation method>
A third embodiment of the present invention is a space forming method for subcutaneously forming a space for subcutaneously administering a protein preparation, which comprises subcutaneously administering chondroitinase. According to this embodiment, even if a low concentration of chondroitinase is administered, the subcutaneous administration restriction amount of the protein preparation can be widened. Further, according to the present embodiment, even if a low concentration of chondroitinase is administered, the transdermal transferability of the protein preparation into the blood can be enhanced.

Details of the chondroitinase and protein preparation are as described in the above embodiment.

In addition, the formation of the space is performed so that the insertion pressure after administration (preferably 2 minutes after administration) is lower than that of the control (protein preparation only), and the insertion pressure is preferably 95% or less of the control. The chondroitinase is administered so as to be preferably 90% or less, more preferably 85% or less. The dose of chondroitinase that achieves the formation of such a space is appropriately determined according to each individual case in consideration of the patient's symptoms, age, sex, and the like.

The effects of the present invention will be described with reference to the following examples and comparative examples. In the examples, the indication of "parts" or "%" may be used, but unless otherwise specified, it indicates "parts by mass" or "% by mass". Unless otherwise specified, each operation is performed at room temperature (25 ° C.).

(Example 1)
The subcutaneous space-forming ability (space-forming ability) of a candidate substance (degrading substance) having an action of decomposing subcutaneous tissue was evaluated.

Insertion pressure when subcutaneously administering a drug to the abdomen of female domestic pigs (17.8-59.0 kg) using pigs, which are said to have similar subcutaneous structure, properties, and thickness to humans, as model animals. Was measured and evaluated.

<Preparation of administered drug>
Water for injection (Nissin Pharmaceutical Co., Ltd.) was added to 0.155 g of L-histidine (Sigma-Aldrich) and 0.760 g of sodium chloride (SIGMA) to make exactly 100 mL, which was used as a solution. Add 60 mL of the lysate to 40 g of Sanglopol (CSL Bering, plasma fractionation preparation (intravenous human immunoglobulin preparation), molecular weight 150,000 Da), dissolve by gently stirring, and add the lysate to make 100 mL, IgG. A solution (protein preparation) was prepared.

12 mL of IgG solution was added to 272.7 mg of hyaluronidase (1100 U / mg, Wako Pure Chemical Industries, Ltd.) and dissolved with gentle stirring at low temperature to prepare a hyaluronidase mixed IgG solution.

Take out 10 powders of chondroitinase ABC (derived from Proteus vulgaris, 10 U / piece, Sigma-Aldrich) into an Eppendorf tube, add 1 mL of water for injection (Nissin Pharmaceutical Co., Ltd.) to dissolve, and dissolve the chondroitinase ABC solution (100 U / piece). mL) was prepared.

<Insert pressure measurement>
The drug to be administered was collected in a syringe. The syringe was connected to the female connector of the extension tube, and the holder of the three-way stopcock was connected to the male connector. Furthermore, the female connector of the winged intravenous needle was connected to the lock adapter of the three-way stopcock, and the transducer was connected to the mixed injection part. After priming, a winged intravenous needle was subcutaneously punctured in the abdomen of the pig. Tissue adhesive was applied around the needle to prevent liquid leakage from the needle hole. According to the administration method shown below, the drug is subcutaneously administered at a rate of 2 mL / min using a syringe pump, and the insertion pressure is measured using the small animal blood pressure measurement system PowerLab from before administration to 5 minutes after the end of administration. did. When the insertion pressure measurement is completed, euthanasia is performed by bleeding from the carotid artery, etc., the tissue at the administration site is collected, fixed with 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd.), and then histopathological examination. Dedicated to. Alcian blue (pH 1.0) (Alcian blue) staining and biotinylated hyaluronic acid-binding protein (Biotin-HABP) staining were performed.

The results are shown in Fig. 2B. FIG. 2A is a micrograph of porcine skin on day 0 after administration of IgG only. As shown in the results, it was confirmed that the light blue-stained chondroitin sulfate disappeared and that chondroitinase ABC could decompose chondroitin sulfate in real time.

<Administration method>
When evaluating the subcutaneous space-forming ability of chondroitinase ABC (Ch ABC), 1 mL of the test drug was administered, followed by 9 mL of the IgG solution, as shown in FIG. When the hyaluronidase mixed IgG solution (HYAL) was administered as a positive control and the IgG solution (IgG) was administered as a solvent control, 10 mL was administered as shown in FIG. 1 below.

The results are shown in Fig. 3. As shown in FIG. 3, it was confirmed that chondroitinase ABC exhibits a space-forming ability equal to or higher than that of hyaluronidase even at a low concentration.

(Example 2)
The ability to form subcutaneous space by dose of the candidate substance was evaluated. The insertion pressure at the time of subcutaneous administration of the drug to the abdomen of female domestic pigs (13.2 to 14.5 kg) in the following group composition was measured and evaluated.

<Preparation of administered drug>
Water for injection (Nissin Pharmaceutical Co., Ltd.) was added to 0.155 g of L-histidine (Sigma-Aldrich) and 0.760 g of sodium chloride (SIGMA) to make exactly 100 mL, which was used as a solution. Add 30 mL of the lysate to 20 g of Sanglopol (CSL Bering, plasma fractionation preparation (intravenous human immunoglobulin preparation), molecular weight 150,000 Da), dissolve by gently stirring, and add the lysate to make 50 mL, IgG. A solution (protein preparation) was prepared.

1.5 mL of the solution was added to 17.442 mg of hyaluronidase (1514 U / mg, derived from Proteus vulgaris, Sigma-Aldrich) and dissolved with gentle stirring at low temperature to prepare a hyaluronidase solution.

Take out 13 powders of chondroitinase ABC (derived from Proteus vulgaris, 10 U / piece, Sigma-Aldrich) into an Eppendorf tube, add 1 mL of water for injection (Nissin Pharmaceutical Co., Ltd.) to dissolve, and dissolve the chondroitinase ABC solution (high concentration). ) Was prepared. Separately, take out 10 powders of chondroitinase ABC (10 U / piece, Sigma-Aldrich) into an Eppendorf tube and add 1 mL of water for injection (Nissin Pharmaceutical Co., Ltd.) to dissolve them to prepare a chondroitinase ABC solution (medium concentration). did. Separately, take out the powder of 5 chondroitinase ABC (10 U / piece, Sigma-Aldrich) into an Eppendorf tube and add 1 mL of water for injection (Nissin Pharmaceutical Co., Ltd.) to dissolve it to prepare a chondroitinase ABC solution (low concentration). did.

<Insert pressure measurement>
The insertion pressure was measured in the same manner as in Example 1.

<Administration method>
1 mL of hyaluronidase solution was added to 9 mL of IgG solution, mixed gently, and 10 mL was administered. The chondroitinase solution of each concentration was also administered in the same manner. As the solvent-controlled IgG, 1 mL of physiological saline was added to 9 mL of the IgG solution, mixed, and 10 mL was administered.

The results are shown in Fig. 4. As shown in FIG. 4, it was confirmed that chondroitinase ABC exhibits a space-forming ability equal to or higher than that of hyaluronidase even at a low concentration. In addition, the lower the concentration of chondroitinase ABC, the lower the insertion pressure. From this result, it is expected that chondroitinase ABC has a high space-forming ability and reaches the threshold of the space-forming ability with a small amount. It should be noted that this consideration is merely an estimate and does not limit the technical scope of the present invention. In FIG. 4, Ch low + IgG is chondroitinase ABC solution (low concentration) + IgG solution, Ch medium + IgG is chondroitinase ABC solution (medium concentration) + IgG solution, and Ch high + IgG is chondroitinase ABC solution. (High concentration) + IgG solution, HYL ++ IgG refers to hyaluronidase solution + IgG solution.

Example 3
Evaluation of pharmacokinetics The blood transferability of subcutaneously administered IgG was evaluated. The drug was subcutaneously administered to the abdomen of female domestic pigs (10 to 15 kg) in the following group composition, and the plasma IgG concentration was measured.

<Preparation of administered drug>
Water for injection (Nissin Pharmaceutical Co., Ltd.) was added to 0.155 g of L-histidine (Sigma-Aldrich) and 0.760 g of sodium chloride (SIGMA) to make exactly 100 mL, which was used as a solution. Add 30 mL of the lysate to 20 g of the antibody drug Sanglopol (CSL Bering, plasma fractionation preparation (intravenous human immunoglobulin preparation), molecular weight 150,000 Da), gently stir to dissolve, and then add the lysate. An IgG solution (protein preparation) was prepared with 50 mL.

1.5 mL of the solution was added to 17.442 mg of hyaluronidase (1500 U / mg, Sigma-Aldrich) and dissolved with gentle stirring at a low temperature to prepare a hyaluronidase solution.

5 powders of chondroitinase ABC (10 U / piece Sigma-Aldrich) were taken out into an Eppendorf tube and dissolved by adding 1 mL of water for injection (Nissin Pharmaceutical Co., Ltd.) to prepare a chondroitinase ABC solution (high 50 U / mL).

<Test method>
Hyaluronidase solution: IgG solution = 1: 9 (volume ratio) was mixed, and 1 mL / kg was administered to the abdomen at 0.2 mL / kg / min. The chondroitinase solution was also administered in the same manner. Venous blood was collected before administration, 5, 10, 30 minutes after the start of administration, 1, 3, 6, 12, 24, 48, 72 hours, and 1 week later using a disposable syringe and a needle. The collected blood was placed in an EDTA / 2K addition container, centrifuged at 3000 rpm at 4 ° C. for 15 minutes, and the obtained plasma was dispensed into a sample tube to prepare a PK measurement sample. The plasma IgG concentration of the PK measurement sample was measured by the ELISA method. _{PK analysis (calculation of AUC, C max} , T _max , t _1/2 ) was performed from the IgG concentration.

The results are shown in Table 5 and Fig. 5 below. In FIG. 5, HYL (1760 U / mL) + IgG refers to a hyaluronidase solution + IgG solution, and Ch (5 U / mL) + IgG refers to a chondroitinase ABC solution + IgG solution.

From the above results, chondroitinase ABC is small, increasing the _{C max} of IgG than hyaluronidase _was shortened _{T max.} This suggests that chondroitinase ABC enhances blood transferability and improves bioavailability compared to hyaluronidase.

This application is based on Japanese Patent Application No. 2019-179812 filed on September 30, 2019, the disclosure of which is referenced and incorporated as a whole.

Claims

A space-forming agent that forms a space under the skin for subcutaneous administration of protein preparations, including chondroitinase.
The space forming agent according to claim 1, wherein the protein which is the active ingredient of the protein preparation has a molecular weight of 10,000 Da or more.
The space-forming agent according to claim 1 or 2, wherein the active ingredient of the protein preparation is an antibody.
The space-forming agent according to any one of claims 1 to 3, which is administered at the same time as the protein preparation.
Subcutaneous administration formulation containing 0.1-100 U / mL chondroitinase and protein formulation.
The subcutaneously administered preparation according to claim 5, wherein the protein which is the active ingredient of the protein preparation has a molecular weight of 10,000 Da or more.
The subcutaneously administered preparation according to claim 5 or 6, wherein the active ingredient of the protein preparation is an antibody.
The subcutaneously administered preparation according to any one of claims 5 to 7, wherein the chondroitinase and the protein preparation are administered at the same time.
A space forming method for subcutaneously administering a protein preparation, which comprises subcutaneously administering chondroitinase.