JPH06336438A - Surface-crosslinked protein prepartion - Google Patents

Abstract

PURPOSE:To provide a surface-crosslinked protein preparation always stably containing a medicinal ingredient and enabling a desired sustained release without deteriorating the original physiological activity of the medicinal ingredient. CONSTITUTION:This preparation is obtained by crosslinking only the surface- near parts of a protein carrier containing a medicinal ingredient, especially a protein medicinal ingredient, with an oleophilic crosslinking agent without affecting the contained medicinal ingredient by the crosslinking reaction, and is stabilized so as to preserve the early physiological activity of the medicinal ingredient as such, achieving a desired sustained release. The surface-crosslinked protein preparation is obtained as follows: adding an aqueous solution comprising the protein carrier and the protein medicinal ingredient to an organic solvent containing a surfactant for stabilizing the emulsion, homogenizing the mixture, pouring the obtained emulsion into a preliminarily cooled organic solvent containing the surfactant to form a protein carrier-containing emulsion, and subsequently adding an oleophilic crosslinking agent (e.g. hexamethylene diisocyanate, oxalyl chloride) to the formed emulsion to crosslink only the surface-near parts of the protein carrier emulsion.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a surface-crosslinking protein preparation using a lipophilic crosslinking agent, and more specifically, using a water-insoluble crosslinking agent, for example, crosslinking only near the surface of emulsion particles containing a protein carrier. Related to the formulation.

[0002]

2. Description of the Related Art In recent years, a sustained-release technique for proteins, physiologically active peptide drugs and the like using a carrier made of a polymer material has been widely studied (CGPitt, Int. J. Pharm., 5
9 , 173 (11990)). As such a carrier for sustained drug release, a material that does not require removal of the carrier from the body even after the drug is sustainedly released, that is, a material having a property of being decomposed and absorbed in a living body is preferable. Thought, now,
Examples of materials having such properties include polylactic acid,
Synthetic polymeric substances such as polyanhydrides and natural polymeric substances such as nucleic acids, polysaccharides and proteins are known. However, in the case of the latter natural polymer substance, since they are water-soluble themselves, it is necessary to prepare a carrier for sustained-release of a drug from these materials in order to prepare water by means of, for example, crosslinking of the materials. Insolubilization is required.

Many water-insolubilization techniques for the above-mentioned natural polymers, particularly proteins, have been reported so far (for example, CA
Finch, in "Chemistry and Technology of Water-Solub
le Polymers ", CAFinch ed. p81, Plenum Press, New
York, 1983, etc.). As a typical example of them, for example, one utilizing protein denaturation by heat treatment (PA
Kramer, J.Pharm.Sci., 63 , 1646 (1974)) and those using chemical crosslinking agents (RCOppenheim ,, Int.J.Pharm., 8,
217 (1981)) and the like. However, when considering inclusion and sustained release of a protein drug by the crosslinked protein carrier obtained as described above,
Deactivation and modification of the included drug by heat or treatment with a chemical crosslinking agent poses a problem. Especially, the chemical cross-linking agents that have been used for cross-linking so far are water-soluble cross-linking agents such as formalin and glutaraldehyde. The major drawback is that the protein drug itself contained in the carrier for the purpose of depolymerization also undergoes a cross-linking reaction, and the sustained-release protein drug is denatured and inactivated, and the sustained release of the drug is not observed due to the cross-linking reaction with the carrier. was there.

More specifically, for example, JP-A-59-462.
Japanese Patent No. 15 describes a method for producing capsules from gelatin, collagen and a mixture thereof, but first of all, it differs from the present invention in that glutaraldehyde is used as a chemical cross-linking agent. Since the drug in this case is a low-molecular weight anticancer drug, sustained release of the drug from gelatin capsules prepared with a water-soluble crosslinking agent is recognized. Also, after the core particles are prepared, a two-step process of coating the core particles with a capsule layer is required,
The capsule preparation method is complicated.

A method for preparing fine gelatin particles containing a protein drug, colony stimulating factor, is disclosed in Japanese Patent Application Laid-Open No. 2-111.
No. 799. In this case, since the fine particles are prepared with a water-soluble chemical cross-linking agent such as glutaraldehyde, the whole gelatin fine particles are cross-linked,
It is considered that the colony stimulating factor contained therein also participates in the cross-linking. Therefore, sustained release of the protein drug is not observed unless the cross-linked gelatin is decomposed.
On the other hand, in the present invention, since only the surface of the gelatin particles is cross-linked, both the gelatin inside the fine particles and the protein drug can be dissolved in water, and if the time is long, it will pass through the surface cross-linked layer of the fine particles. , Sustained release in vitro. In fact, unlike the glutaraldehyde-crosslinked gelatin fine particles, the surface-crosslinked gelatin particles of the present invention showed sustained release of the protein drug. This is because the lipophilic chemical cross-linking agent was used to cross-link only the vicinity of the surface of the particles, and this point is greatly different.

On the other hand, Japanese Patent Application Laid-Open No. 60-100516 discloses a method for producing sustained-release microcapsules. Among them, a protein such as gelatin is mentioned as a microcapsule material, which is used as a core substance of the microcapsule. Further, as a material for encapsulating the core microspheres, a synthetic polymer substance that is sparingly soluble or insoluble in water is used, which is basically different from the present invention.

[0007]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to eliminate the drawbacks found in the conventional use of a cross-linking type carrier for sustained release of a protein drug of this kind, and to solve the original physiological problem of the drug. It is an object of the present invention to provide a new preparation capable of always stably incorporating the drug and achieving its desired sustained release without any activity reduction or inactivation at all.

[0008]

Means for Solving the Problems As a result of intensive studies conducted by the present inventor for the above purpose, when a lipophilic chemical cross-linking agent is used, the protein carrier is cross-linked only near the surface,
It is possible to prepare a bag-shaped protein preparation in which only the surface is cross-linked, whereby the drug present in the bag is not subjected to any cross-linking reaction, and the initial physiological activity is maintained as it is and stabilized. Thus, the fact that the desired sustained-release preparation was obtained was found, and the present invention was completed here.

That is, the present invention provides a surface-crosslinking protein preparation using a lipophilic crosslinking agent, and a surface-crosslinking protein preparation characterized in that a protein carrier including a protein drug is surface-crosslinked with the lipophilic crosslinking agent. The manufacturing method of

By using the method of the present invention, the surface-crosslinked protein preparation of the present invention can produce capsule-type microspheres in which only the vicinity of the surface of the protein carrier is crosslinked by using the method of the present invention. For example, the surface-crosslinked protein preparation containing the protein drug of the present invention can be obtained by the following method.

That is, a protein carrier and an organic solvent containing a surfactant for stabilizing the emulsion, such as a mixed solvent of toluene, chloroform, cyclohexane, or the like, or a mineral oil such as olive oil or cottonseed oil, is used. Add an aqueous solution of protein drug. The protein carrier concentration at that time is usually preferably 5 to 30%, and the amount of the protein drug with respect to the amount is preferably about 30% by weight or less. The emulsion obtained by homogenizing the mixture is rapidly poured into a pre-cooled organic solvent or mineral oil containing a surfactant to form a protein carrier emulsion containing a protein drug. At this time, the volume ratio of the aqueous protein solution to the organic solvent or mineral oil is preferably in the range of about 1/5 to 1/20. Then, a lipophilic cross-linking agent is added to this emulsion, whereby only the vicinity of the surface of the protein carrier emulsion particles can be cross-linked. The amount of the cross-linking agent added is preferably about 1 to 10.times.10@-4 mmol per mg of the protein carrier. After centrifugation, the supernatant is discarded, and the precipitate (surface-crosslinked protein preparation) is washed with an organic solvent and a phosphate buffer solution (pH 7.4) by centrifugation and lyophilized to obtain a drug-containing surface-crosslinked protein preparation. .

The type of the lipophilic cross-linking agent used in the present invention is not particularly limited, but it has a high reactivity with protein molecules for the purpose of relaxing the conditions of the cross-linking reaction such as cross-linking time and temperature as much as possible. Functional crosslinkers are preferred.
Possible functional groups that react instantaneously with amino groups, hydroxyl groups, etc. in protein molecules include chlorides of isocyanate groups and carboxyl groups, etc., and any parent having two such functional groups in one molecule. Oily crosslinkers can be used. Specific examples thereof include hexamethylene diisocyanate, hexamethylene diisocyanate trimethylolpropane adduct, hexamethylene diisocyanate isocyanurate, toluene-2,4-diisocyanate, oxalyl chloride, succinyl chloride, adipoyl chloride, terephthaloyl chloride. Etc. can be illustrated. In addition to the above, it is also possible to use, for example, a lipophilic dicyclohexylcarbodiimide which catalyzes the reaction between an amino group and a carboxyl group in a protein molecule.

Examples of the surfactant include sorbitan monooleate (Span 80, manufactured by Wako Pure Chemical Industries, Ltd.), sorbitan trioleate (Span 85 (Spa 85)).
n 85), Wako Pure Chemical Industries, Ltd.) and the like nonionic surfactants are used.

The type of protein used as a drug carrier is not particularly limited, and the surface cross-linking method of the present invention is applicable to all types of proteins such as gelatin, collagen, albumin and the like.

In the present invention, the protein drug is
There is no particular limitation, and growth factors, peptide drugs such as insulin, as well as high-molecular weight compounds such as interferon, tumor necrosis factor, lymphotoxin, interleukins, various growth factors, superoxide dismutase, therapeutic proteases, etc. All types of drugs such as protein drugs are available.

The particle size of the preparation of the present invention can be controlled by the strength of homogenization, time and the like. Examples of the homogenization method include a method using a homogenizer, an ultrasonic emulsification method, a microfluidizer method, and the like.
It is possible to reduce it to 0 nm.

Thus, according to the above method, it is possible to prepare a bag-shaped protein preparation in which only the vicinity of the surface is cross-linked, which could not be obtained by the conventional use of a water-soluble chemical cross-linking agent. The preparation is in an aqueous solution state in which the protein inside is not crosslinked. The use of the formulation of the present invention can be variously considered depending on its shape,
For example, when considering the sustained release of a protein drug, the included drug does not undergo inactivation due to cross-linking, and the sustained release of the drug in a physiologically active state is possible.

The protein preparations described above can be used by dispersing them in a phosphate buffer solution (pH 7.4), a solvent for injection such as physiological saline or a diluent, but they are dried and dispersed in the diluent at the time of use. May be used.

The surface-crosslinked protein preparation of the present invention thus obtained is a bag-shaped protein preparation in which only the surface is crosslinked, and the protein or peptide drug is not subjected to a crosslinking reaction in the bag and is in its original state. As a result, a drug having physiological activity is sustainedly released, and there is no fear of inactivation or deterioration of sustained release property.

[0020]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0021]

Example 1 4 ml of chloroform and 6 m of cyclohexane
sorbitan trioleate (Span
85, 100 mg of Wako Pure Chemical Industries, Ltd. was dissolved, and 20 mg of gelatin (alkali type, pI 4.9, manufactured by Nitta Gelatin Co., Ltd.) and bovine serum albumin (B
SA) 2 mg containing 0.4 M carbonate buffer solution (p
H9.8) 0.2 ml was added and the mixture was emulsified using a homogenizer.

The resulting emulsion was quickly added to 10 ml of the previously mixed chloroform / cyclohexane mixed organic solution containing the Span 85 that had been previously cooled. Then, 2.5 ml of the above chloroform / cyclohexane (4: 6) mixed solution containing succinyl chloride at a concentration of 1.9 × 10 ⁻⁴ mmol / mg gelatin was added thereto, and the mixture was added at 0 ° C.
And stirred for 3 minutes. After the reaction, the emulsion was centrifuged (4000 rpm, 5 minutes), and the supernatant was discarded. Precipitation part (BSA-containing surface cross-linked gelatin microspheres)
Was washed with the above-mentioned chloroform / cyclohexane (4: 6) mixed solution by centrifugation 3 times, and then washed with a phosphate buffered saline solution (PBS, pH 7.4) 3 times to obtain the desired microspheres. Got

The result of the optical micrograph of the obtained BSA-containing surface-crosslinked gelatin microspheres in PBS is shown as A in FIG. Incidentally, B in FIG. 1 is a BSA-containing gelatin microsphere prepared by using glutaraldehyde as a water-soluble crosslinking agent for comparison (Japanese Patent Laid-Open No. 2-1.
No. 11799).

From the figure, it is understood that according to the present embodiment, capsule-type microspheres having a bag-like structure are prepared.

[0025]

Example 2 Chloroform 4 ml and cyclohexane 6 m
sorbitan trioleate (Span
85, Wako Pure Chemical Industries, Ltd.) (100 mg) was dissolved, and 0.4 M carbonate buffer solution (pH 9.8) containing gelatin (alkali type, pI 4.9, Nitta Gelatin Co., Ltd.) and BSA in this solution was added. 2 ml was added and the mixture was emulsified using a homogenizer. The amount of gelatin added was 20, 40 and 60 mg respectively, and BSA was added.
Is 10% by weight with respect to gelatin, ie 2,
4 and 6 mg respectively.

The resulting emulsion was quickly added to 10 ml of the previously cooled chloroform / cyclohexane (4: 6) mixed organic solution containing Span 85. Then, the above chloroform / cyclohexane mixed solution containing various concentrations of succinyl chloride.
5 ml was added, and the mixture was stirred at 0 ° C. for 3 minutes. After the reaction, the emulsion was centrifuged (4000 rpm, 5 minutes), and the supernatant was discarded. The precipitated portion (BSA-containing surface-crosslinked gelatin microspheres) was washed with the above chloroform / cyclohexane (4: 6) mixed solution by centrifugation three times, and then with a phosphate buffered saline solution (PBS, pH 7.4). Centrifugal washing was repeated 3 times and freeze-dried to obtain the desired microspheres.

The yield and BSA content of the obtained BSA-containing surface-crosslinked gelatin microspheres are shown in Table 1 together with the concentrations of gelatin and succinyl chloride used. The above-mentioned yield and BSA content were examined using the weight ratio before and after preparation of microspheres and ¹²⁵ I-labeled BSA, respectively.

[0028]

[Table 1] From the table, it can be seen that the yield of microspheres increases with an increase in the amount of gelatin fed. This indicates that when the concentration of gelatin is low, the crosslinking reaction does not proceed well and few surface-crosslinked microspheres are obtained. In addition, their yields are almost the same as the BSA content of the corresponding microspheres, and it is considered that BSA is contained in the microspheres in almost 100%.

[0029]

Example 3 Surface-crosslinking gelatin microspheres containing TNF were prepared in the same manner as in Example 2 except that human gene recombinant tumor necrosis factor (TNF, manufactured by Dainippon Pharmaceutical Co., Ltd.) was used instead of BSA. Was prepared.

The amount of TNF charged was 2.5 × 10 ³ units / mg gelatin.

The yield and TNF content of the obtained TNF-containing gelatin microspheres were determined in the same manner as in Example 1 and the results are shown in Table 2.

[0032]

[Table 2] Also in this example, as in Example 1, TNF was contained in the microspheres at a content rate of almost 100%.

[0033]

Example 4 The in vitro degradability of the BSA-containing surface-crosslinked gelatin microspheres prepared in Example 2 was evaluated in collagen-containing (8 × 10 ⁻⁴ mg / ml) or PBS without collagenase. It was evaluated by measuring the protein amount of the remaining microspheres after shaking at 37 ° C. by the ninhydrin method.

The results are shown in FIG.

From FIG. 2, it can be seen that the surface-crosslinked gelatin microspheres decompose over time. It is also understood that the degradability can be controlled by the gelatin concentration at the time of preparing the microsphere. The addition of collagenase accelerated the decomposition of microspheres.

On the other hand, in the degradability test of microspheres in PBS containing no collagenase, the microspheres after 4 days were observed with an optical microscope to find that the degradation was 30
Almost the same number of capsule-type microspheres remained after the minute. It is considered that this is because the gelatin capsule was not decomposed in PBS and the gelatin molecules in the capsule were eluted by diffusion through the capsule membrane.

[0037]

Example 5 In vitro sustained release of TNF from TNF-containing surface-crosslinked gelatin microspheres prepared in the same manner as in Example 3 using ¹²⁵ I-labeled TNF,
The microspheres were examined by measuring ¹²⁵ I radioactivity in PBS after shaking for a specified time at 37 ° C.

The results are shown in FIG.

From FIG. 3, it can be seen that TNF is gradually released from each microsphere with time. It is also found that the sustained release pattern can be controlled by the gelatin concentration during the production of microspheres.

[0040]

Example 6 Viable cells after culturing mouse fibrosarcoma (MethA) for 24 hours with TNF contained in surface cross-linked gelatin microspheres, TNF not contained and empty microspheres, respectively. I checked the number. As a control, cells were grown in a normal state without adding anything to the culture system.

The cell growth inhibition rate was calculated from the ratio of the number of each viable cell to that of the control group. It is indicated that the larger this value is, the larger the growth inhibitory activity is.

The results are shown in FIG.

From FIG. 4, cell proliferation was suppressed in a TNF concentration-dependent manner in the presence of TNF, regardless of inclusion in microspheres. However, inclusion in microspheres shows the same growth inhibitory activity with a smaller amount of TNF compared to free TNF. This indicates that the sustained release of TNF effectively acts on the expression of its physiological activity.

[Brief description of drawings]

1 is an optical micrograph (A) as a substitute for a drawing showing the particle structure of BSA-containing surface-crosslinked gelatin microspheres obtained in Example 1 in PBS, and prepared by using glutaraldehyde as a water-soluble crosslinking agent. It is the same photograph (B) of the prepared BSA-containing gelatin microspheres.

FIG. 2 is a graph showing the in vitro degradability of the BSA-containing surface-crosslinked gelatin microspheres produced in Example 2.

FIG. 3 is a graph showing the sustained release of TNF from TNF-containing surface-crosslinked gelatin microspheres in vitro by measuring ¹²⁵ I radioactivity.

FIG. 4 is a graph evaluating the cell growth inhibitory effect of mouse fibrosarcoma cells by TNF contained in surface-crosslinked gelatin microspheres.

Claims (2)

[Claims] 1. A surface cross-linking protein preparation using a lipophilic cross-linking agent. 2. The method for producing a surface-crosslinking protein preparation according to claim 1, wherein a protein carrier including a protein drug is surface-crosslinked with a lipophilic crosslinking agent.