JPH0249734A - Novel composition - Google Patents

Abstract

PURPOSE:To obtain a composition suitable for organism and effective in the field of medicine or cosmetic, etc., having improved stability of growth factor, etc., with low antigenicity and toxicity, containing growth factor, etc., and gelatins as active ingredients. CONSTITUTION:At least (A) growth factor, peptide corresponding to a part of the constituent of said growth factor, derivative of the above compound or salt of the above compound and (B) gelatins are mixed and contained in the aimed composition as active ingredients. Growth factor group of epithelial cell such as growth factor of epithelial cell or transforming growth factors, etc., insulin group or growth factor group of cell derived from platelet, etc., is used as a growth factor and prepared by the method of extraction from organism (e.g., blood, salivary juice or urine) or chemical synthesis, etc. Besides, in use, 1-1000 microgram/l concentration of the growth factor and 0.1-10mg/l, concentration of gelatins as stabilizer are preferable in the case of injection.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION TECHNICAL FIELD This invention relates to novel compositions. More specifically, the present invention relates to growth factors, peptides corresponding to some of their constituent components, derivatives thereof, or salts thereof (hereinafter sometimes referred to as "growth factors, etc."), and gelatins. The present invention relates to a composition comprising at least

Prior art growth factor (Growth f'actor) is ri
``a substance that promotes the growth of animal cells in vivo or in vitro and is not a nutritional substance'' (Ann, Rev. Biochem, 45
．． 531-558 (197B)), conventional hormones and their carriers also fall under the category of growth factors.

Approximately 40 types of such factors are currently known, and the main growth factors include those classified into the insulin family (insulin, insulin-like growth factor ((IGF-
I, ICF-II, etc.), mammary gland stimulating factor (M SF )
), nerve growth factor ((NG
)),) Transforming growth factors (TGF,
TGFβ, TGF-A, etc.), α Those classified into the platelet-derived growth factor family (platelet-derived growth factor (PDGF)), osteosarcoma-derived growth factor (ODGF)), fibroblast growth factor (F G
F)), etc.), others (colony formation stimulating factor <<
CSF, )), T cell growth factor, tumor angiogenesis factor (
(TAF)), DNA synthesis promoting factor (DSF)
)), tumor-derived cell growth factors, fibroblast-derived growth factors (FDGF), etc.).

Among these growth factors, epidermal growth factor has been isolated from the urine of humans and horses, and from the submandibular glands of rabbits, rats, and mice, and is present in mammals across species. Known 1: Adv, Metab, D
is,, 8.285 (1975), JP-A-56-25
Publication No. 112, etc.]. Among them, human epidermal growth factor (
human Epidermal Growth Fa
ctor:hEGF) was developed in 1975 by Cohen (S.
It was introduced by Cohen et al. as a human-derived factor that promotes the proliferation and keratinization of epithelial tissue isolated from human urine (
Proc.

Na11. ^cad, Set, USA, 72.1
317 (1975)), and Gregory (H,G) in the same year.
Human urogastron (human U), which has the effect of suppressing gastric acid secretion, was isolated from human urine by
rogastrone: h-UG) was introduced as (
Nature, 257.325 (1975)) is the same substance as the polypeptide, and has a molecular weight of about 6000 and 53
A polypeptide consisting of amino acid residues and having three disulfide bonds in its molecule [Metabolism, 17.5
1-5g (1980)) (hereinafter, epidermal growth factor will be referred to as EGF).

The physiological and pharmacological activities of EGF that have been reported to date include its gastric acid secretion suppressing effect (Gut).
, 18.1877 (1975), 1dld, , 23,
951 (1982)), anti-ulcer effect (Gut, 22.
927 (1981), Br1d.

J, Surg, [i4,830 (1977)), Gastrointestinal mucosal protective effect [JP-A-60-9686],
DNA synthesis promoting effect (Gut, 22,927 (1981
), J, Physjol,, 325.

35 (1982)), Corneal repair action [JP-A-59-6
No. 5020], calcium release promotion effect (Endo
crinology, 107.270 (1980))
, wound healing promoting effect (Plast, Rlconstr
, Surg., 64.766 (1979), J.
Surg, Res, 33, 164 (1982))
, anti-inflammatory action (Japanese Unexamined Patent Publication No. 60-115784), and analgesic action (Japanese Unexamined Patent Publication No. 60-115785).

However, since EGF is unstable, it cannot be stored for a long period of time, and it has not yet been commercialized.

Therefore, attempts were made to introduce one or more polymers such as polyoxyethylene-polyoxypropylene copolymer into the EGF molecule to stabilize it, but although the stability was improved, the physiology of EGF itself It has not been put into practical use due to the decreased activity (Japanese Patent Application Laid-open Nos. 59-59629 and 58225025).

In addition, by blending EGF with polysaccharides such as cellulose derivatives, starch, agar, alginic acid, and gum arabic,
Although the stability was greatly improved (Japanese Patent Application Laid-Open No. 63-15232
4), other stabilization methods are desired due to the need for further improvement in stability, poor storage stability of polysaccharides, and high antigenicity.

[Summary of the invention]

Summary The present invention aims to solve the above problems, and as a result of extensive research, it has been discovered that the stability of growth factors, etc. can be improved by combining them with gelatin. The aim is to achieve the above object by completing a new composition based on the knowledge and providing it.

Therefore, the composition according to the present invention comprises at least (a) a growth factor, a peptide corresponding to a part of its constituent components, a derivative thereof, or a salt thereof, and (b) a gelatin. This is a characteristic feature.

Effects The novel composition according to the present invention comprises at least the above-mentioned two components, that is, a growth factor, a peptide corresponding to a part of its constituent components, a derivative thereof, or a salt thereof, and gelatin. This composition provides excellent stability, low antigenicity and toxicity, and is suitable for living organisms.

It is understood that the stabilizing effect of gelatin on growth factors and the like was unexpected to those skilled in the art. Needless to say, the composition according to the present invention makes a significant contribution in the fields of pharmaceuticals, cosmetics, etc.

[Detailed description of the invention]

Growth factors, etc. (component (a)) In the present invention, "growth factors" refer to in vivo or 10
As mentioned above, VttrO refers to substances that promote the growth of animal cells and are not nutritional substances, and conventional hormones and their carriers are also included in the category of growth factors.

As mentioned above, growth factors include the epidermal growth factor family,
There are insulin family, platelet-derived growth factor family, etc., all of which are subject to the present invention, but among these, representative ones are those of the epidermal growth factor family, such as epidermal growth factor (EGF) and transforming growth factor. factors (TGF) and insulin family members such as insulin and insulin-like growth factors (IGF-1, IGF-
11) and members of the platelet-derived cell growth factor family, such as fibroblast growth factor (FGF).

The present invention relates not only to the above-mentioned naturally occurring growth factors, but also to growth factors that are structurally different from the above-mentioned factors in terms of arbitrary substitutions, additions or deletions of amino acids constituting the growth factors, etc. It also includes fragments and derivatives that have the same or similar physiological and pharmacological activities. Note that the amino acid that can be arbitrarily substituted as a derivative may be any amino acid as long as it has the activity of a growth factor.
The amino acid to be substituted may be a natural one or a derivative (analog (for example, one with fluorescence, one with high fat solubility, etc.)).

Further, the growth factor derivatives also include fragment derivatives. A fragment may be a portion that has at least binding activity to a growth factor receptor, and has a partial structure of a part of the growth factor. For example, one or more amino acids are missing from the C-terminus or N-terminus, or
It includes those formed by cleavage from the terminal or any arbitrary part and consists of two or more amino acids.

In addition, among the above factors and their derivatives mentioned above, derivatives that have not lost their terminal amino and carboxyl groups through chemical modification have a-distant amino and carboxyl groups, so salts with acids and It can also be a salt with a base. In that case, pharmaceutically acceptable organic or inorganic acids and bases can generally be used, such as hydrochloric acid, sulfuric acid, acetic acid, malonic acid, succinic acid, sodium hydroxide, amines, etc. can be mentioned.

Derivatives also include those subjected to various chemical modifications. Such derivatives include, for example, AlA
It can be obtained by chemical modification known per se such as ylation, oxidation, reduction, ice-melting, or a combination thereof.

Therefore, the present invention provides not only the above-mentioned growth factors themselves, but also
It also includes peptides (fragments) having partial structures thereof, derivatives thereof, and salts thereof.

In addition, these growth factors etc. are used in living organisms (specifically, blood,
(Growth factors and their derivatives, etc. can be extracted from saliva, urine, lachrymal fluid, breast milk, various tissues/organs, etc.) or prepared by various methods [edited by the Japanese Society of Tissue Culture [Cell Growth Factor J]
(Refer to the literature included in Asakura Shoten, 1984)), chemical synthesis, or genetic engineering synthesis techniques. The growth factors etc. prepared in this way are preferably of high purity.

Epidermal cell growth factor is a typical example of such growth factors. As mentioned above, epidermal growth factor has been isolated from the urine of humans and horses, as well as from the submandibular gland of rabbits, rats, and mice, and is present in mammalian animals across species. is known (Adv, Met
ab, Dis, 8.265 (1975), JP-A-56-25112, etc.]. Among these, when the drug of the present invention is applied to humans, the hEGF is preferred. To prepare this EGF, for example, a method of isolating it from biological components [JP-A-58-99418, JP-A-58-2191]
No. 24, Publication No. 59-2041.23, etc., Special Publication No. 1973
-12744, 53-4527, 59-503
No. 15, No. 59-50316, No. 59-42650, etc.] and chemical synthesis method [Unexamined Japanese Patent Publication No. 59-278]
58 Publication etc.] and the method of creating it by genetic engineering method [JP-A-57-122096, JP-A No. 58-2166
No. 97, Publication No. 59-42650, etc.] have been proposed.

On the other hand, EGF derivatives are those created by genetic engineering methods [human EGF (hEGF), in which leucine is substituted for methionine at the 21st position from the N-terminus of the 53 amino acid residues ((Le u''' )-hE
GF: Japanese Unexamined Patent Publication No. 60-28994)] has been proposed. As another EGF derivative, hEGF-II, which is hEGF lacking two C-termini, has also been obtained [
Patent application proposed by the present inventors and co-researchers
See specification of No. 0-22630. Here, the h
EGF has high purity (approximately 99.9% or more), and such high purity is preferable as component (a). ]. Still other EGF fragments: EG
des-, which is F with 5 or 6 missing C-terminals
(4953)-EGF (EGF-5), des
-(48-53) -EGF (EGF -6)
(Bioch-emistry, 15.2624(19
7B), Mo1. Pharmacol,, 17゜3
14 (1980), Vlatm, 11orm, 37
．． 89 (1979), Cl1n.

Res, 25, 312A (1977)), E
EGF (20-31), which is considered to be the minimum unit for GF to express its various effects, and its derivative C (
Acm)Cys)EGF-(20-31)
[Proc.

20.31 Natl, Aead, Sci, 81.1351 (1
9g4) and [Ala20]EGF-(14-31)
[Proc, Natl, Acad, Sci.

81.1351 (1984)), CNBr-, a derivative obtained by treatment with cyanogen bromide.
E G F (131 oche I Wistry, 15
,2[124(197B):1 etc. are also known,
Such substances also fall within the scope of growth factors, etc. of the present invention.

One group of growth factors is the epidermal growth factor family, examples of which are the above-mentioned EGF and transforming growth factors.
Fae-Lor: TGr') has been in existence for five years.

This TGF has large similarities in amino acid sequence with EGF, and is currently classified into three classes. That is, TGF.

α TGFβ and TGF7. TGFβ is TGFa
Alternatively, it acts synergistically with EGF to promote cell growth. TGF alone exerts this effect γ. EGF and these three TGFs belong to the same growth factor family, and TGF in particular has a high amino acid sequence similarity to EGFα, with up to 21 of the 50α residues of TGF being found at homologous positions in EGF. (Proc, Natl, Acad, Sc1.lN,
7363 (1984)). In addition, TGF α binds to the EGF receptor, which competes with EGF [“The History of Medicine” 1
33.1040-1044 (1985)).

Furthermore, TGF activates the EGF receptor kinase with αNature, 292.259<1981
)), anti-EGF receptor antibody blocks the vulacrine action of TGF (J, B[ol, Ch-em, 258.1189
5 (1984)), from these facts, EGF and TG
It is understood that F has a very similar effect. Similarly, insulin and IGFs, TGF and EG
It has been reported that there is a similar effect, such as the relationship α with F [
Adv, Metab, Dis, 8.203,211
, 237 (1975), Endocrinology,
107.1451 (1980) ) o and IGF
In addition to their insulin-like activity, they also have growth factor activity. The present invention also includes homologous factors having very similar effects as these.

Gelatins Gelatins in the present invention may be a series of induced proteins obtained by treating gelatin, that is collagen, with hot water, or may be a series of induced proteins obtained by treating these induced proteins with acid or alkali, or by alkyl or halogenation, etc. It may also be one that has been subjected to physicochemical treatment to the extent that it does not significantly change.

This gelatin is preferably one obtained by treating the above-mentioned induced protein with an acid or alkali as shown in the experimental example below, or a low molecular weight (water-soluble) gelatin (molecular ffi: 5,000
0 to 10,000). All of these gelatins have low toxicity as they are often used as additives in medical products or food additives (for the definitions of gelatin and collagen, see the Chemical Encyclopedia (published by Kyoritsu Publishing Co., Ltd.) ).

Composition The composition according to the present invention contains at least the above-mentioned growth factors and gelatin, and can be in any form. For example, if the composition is for pharmaceutical use, it can be in any form that can be administered orally or parenterally, and if it is for cosmetics, it can be in any form suitable for skin application or other forms suitable for that purpose. It can be.

Specific examples of dosage forms for such compositions include oral solutions, syrups, injections, powders, granules, tablets, emulsions,
There are paste preparations, poultices, liniments, half-split preparations, aerosol preparations, ophthalmic solutions, plasters, and ointments (including creams, gels, and eye ointments), etc. (including preparations). It goes without saying that these dosage forms can be appropriately selected depending on the therapeutic purpose. A preferred example of a drug according to the present invention is a dosage form that can be applied to an injection as shown in the experimental examples below. More preferred are compositions for the ready-to-use preparation of these drugs. The method for preparing this composition is, for example, by suspending gelatin in a solvent that does not contain growth factors, etc., stirring and dissolving the mixture while heating to prevent bubbles as much as possible, and cooling to room temperature. It can be obtained by adding, stirring, dissolving, and freeze-drying.

In addition, pharmaceutically acceptable preservatives, soothing agents, buffers, surfactants, etc. may be present in the composition as required.

The composition of the present invention varies depending on the purpose of use or dosage form, but
When used as an injection, the concentration of growth factors etc. is 1 to 1.
000 micrograms/ml, and gelatin as a stabilizer is preferably 0.1 to 10 mg/ml.
A range of is preferred.

The dosage of the composition of the present invention as a medicine should be based on known levels of growth factors, etc., and should be such that the growth factors, etc. in the composition reach such levels. Can be done.

Experimental Example Stability Test A study was conducted to determine whether h-EGF compositions containing gelatin have a significant effect on stabilizing h-EGF.

How to get there Samples were prepared using the following recipe.

(1) 4mg Gelatin (acid or alkali treatment) 1mg h-EGF PBS (pH7,2) 0.02+ng Tween80 (++) 5mg Human serum albumin 23mg
Glycine 1 mg h -E G F PBS (pH 7,2) 0, 02 mg Tween 80 (iil) 5 mg MC, HPMC or CMC
N al mg h -E G F PBS (pH 7,2) 0, 02 mg Tween 80 Note) PBS diphosphate buffer MC: Methylcellulose HPMC: Hydroquineprovil methylcellulose CMCNa: Sodium carboxymethylcellulose Mix these by an appropriate method. After dissolving in 2ml of water, 0.4
ml was dispensed into 10 ml vials and freeze-dried. Then, after half a month, a month, and two months, it was stored in a constant temperature room at 35°C.
0.4 ml of water was added to the vial, and the h-EGF residual rate was calculated by high performance liquid chromatography (HPLC).

The measurement method is more quantitative than the receptor binding analysis method, so H
PLC method was adopted. Details of the HPLC method are as follows.

Conditions: JASCO Intelligent HPLC System 800, liquid chromatography column with multi-wavelength detector: TOYO5ODA TSK-GEL DDS120
T, 250X4.8mm Eluent A: 1% acetonitrile, 0.05% trifluoroacetic acid, 0
．． 025% triethylamine (remainder water for liquid chromatography) Eluent B: 80% acetonitrile, 0.05% trifluoroacetic acid,
0.025% triethylamine (the rest is water for liquid chromatography) Ratio of A and B: A70%-B30% (t-0 min) to A60%-840
% (t-10 min).

Flow rate: 1 ml/min Column temperature: +40°C Sample solution: Add a certain amount of water to the lyophilized composition containing a certain amount of h-EGF in a vial to dissolve it, and use a certain amount of this solution for chromatography. there was.

The residual rate of h-EGF in the sample was calculated according to the following formula.

Results〉 The results are shown in the table below. It can be said that gelatins significantly suppressed the decomposition of h-EGF compared to sugars (celluloses).

Type of additive (%) After 2 months 78.8 89.7 88.8 81.1 84.9 83.6 83.0 Residual rate of h-EGF After half a month After 1 month 90.2 82.9 92.9 91.0 90.7 91.5 88.8 8f'i, 8 88.9 86.0 8g, 1 84.0 89.7 88.5 Acid-treated gelatin Alkali-treated gelatin Human serum albumin C PMC MCNa Consideration of Formulation Although the growth factors and the like used in the present invention can be mass-produced through genetic engineering techniques, contamination with endotoxin due to this has become a major problem in formulation, particularly in the production of injections. Therefore, we investigated the formulation of a stable injection, including the removal of endotoxin from h-EGF.

a. Pyrogen removal First, various filtration membranes, such as microfiltration membranes (product name: M
ILLEX-GV membrane) and a charge-modified Depson filtration membrane (trade name: Disposable, Zetapore membrane filter) were used to investigate pyrodiene removal. The principle is that in the case of microfiltration membranes, the polymer type is polyvinylidene difluoride, which is expected to form a hydrophobic bond with endotoxin. Furthermore, in the case of a charged depth filtration membrane, an ionic bond between the ten charges of nylon 66 containing a quaternary ringinium group and the one charge of endotoxin is expected. the result,
For microfiltration membranes, endotoxin (10 ng/ml, E, col! UK) in PBS, pH 7,2 solution.
(purified from cultured cells of strain T-8) could not be removed and had no effect. In the case of a charged Debsen filtration membrane carried out in the same manner, the amount of endotoxin in the liquid after filtration was 0.1.95% g/
ml t, was not present, resulting in a reduction of endotoxin to approximately 1150. However, h −EG
Since the recovery rate of F (0.5 mg/ml) was 77%, it is clear that this method cannot be used. Ta.

Note that endotoxin was quantified using the Limulus method. h
-EGF was quantified using HPLC.

Next, the following study was conducted using an affinity resin (trade name: Virosep 0) in which histidine was covalently bonded to Se, Awayu 0 using xamethylenediamine as a spacer.

A 1×10 cm column filled with the above Pyrosep was prepared and tested with buffer solutions of various pH and salt concentrations.

Conditions are flow rate +6 ml/h, fraction amount: 2 ml
% performed at room temperature. To use Pyrosep■ in a column, suspend Pyrosep■ in pyrogen-free water,
It was packed in a column and equilibrated with a solvent capable of dissolving the sample in an amount more than three times the adsorbent capacity. As a result, the pH of the phosphate buffer solution, which is commonly used as a raw material for pharmaceuticals, was around neutral, and the salt concentration (20mM NaHPO・2H20,46,7
mMNa2HPo 12H20) was a suitable eluent.

It has become clear that if the salt concentration is higher than this, adsorption of endotoxin is poor, and if the salt concentration is lower than this, h-EGF is adsorbed.

Based on these results, a sample solution (0.5 mg/ml h-EGF, 20 mM Na
HP O fist 2H20-46, 7mMNa2HPO4・1
2H20, pH 7, 2, 2ng/ml endotoxin)
was prepared, and the amount of endotoxin and h-EGF in this sample solution after passing through the column was measured. The conditions are as described above. The results are shown in the table below. EGF
It was possible to selectively remove endotoxin without adsorbing it.

Result fraction number Endotoxinffi h-EG
F recovery rate (ng/ml) (%) 9 <0. 1 9610
<0. 1 10011
<0.1 10015 <0
．． 1 10020 <0. 1
100b, Manufacture of injections Under aseptic conditions, the aqueous solution of component A below was treated with an ultrafiltration membrane, and then the aqueous solution of component B below was filled with Pyrosep and subjected to a column to remove pyrodiene. processed.

These image processing solutions were mixed and lyophilized to prepare an injection ready for use. In addition, this preparation shall be used by adding 2 ml of distilled water for injection at the time of use.

Ingredient A Gelatin 4. Oa+g Tveen 80 0. 02+ng Component B -EGF NaH2PO4' 2JO Na2HPO4・12H20 1.0mg 6.4+ng 33.5 tai g

Claims (1)

[Claims] A composition comprising at least (a) a growth factor, a peptide corresponding to a part of its constituent components, a derivative thereof, or a salt thereof, and (b) gelatin.