JP2005082584A - Method for using recombinant gelatinous protein as plasma augmentor and composition suitable as substitute for plasma - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a composition containing a plasma augmentor having a low clearance speed in a blood circulation system and capable of decreasing the occurrence of immune reactions, especially, anaphylactic shock, and therefore suitable as a substitute for plasma. <P>SOLUTION: The composition contains a human recombinant gelatinous protein as the plasma augmentor, wherein the gelatinous protein has a molecular weight of at least 10,000 dalton and at most 50,000 dalton and an isoelectric point of less than 8. The gelatinous protein may comprise a polymer, such as a dimer, a trimer, and a tetramer. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to the use of a recombinant gelatin-like protein (or polypeptide) as a plasma expander and a composition comprising such a plasma expander and suitable as a substitute plasma.

  Established as a method of using gelatin is a method of using blood plasma as a colloidal solution. Such plasma substitutes can be used to control circulatory blood flow, for example, to cope with shocks such as urgent bleeding or burns. It should be noted that the gelatin solution is made sterile, pyrogen-free and antigen-free and that the molecular size can be averaged to maintain the desired colloid osmotic pressure. In order to maintain a sufficient colloid osmotic pressure to obtain a sufficient amount of blood circulation and to obtain a blood pressure that is sufficiently effective over a suitable period of time, the size of the gelatin molecule is made less prone to gelation.

  In order to make gelatin suitable as a plasma extender, chemical modification has been performed for the purpose of significantly reducing gelling ability. For this purpose, it is known to crosslink gelatin simultaneously with degradation, to form branched or intramolecular bridges from gelatin molecules. The most successful modification is probably the succinylated gelatin preparation described in US Pat. No. 2,827,419. A commercial preparation based on succinylated gelatin is known as Gelofusine and is currently commercially available. The gelatin used for this is isolated from bovine-derived gelatin and has an average molecular weight of 30,000. In addition, Geloplasma ("polygelatin") and Gerifundol ("oxypolygelatin") are commercially available as modified gelatin.

  WO 01/34646 A2 describes a recombinant gelatin structure with various molecular weights and various stages of hydroxylation. Among the many possible uses of recombinant gelatin in the claims, there is a use as a plasma expander. However, in the above invention, there is no definition of a structure suitable for a plasma extender, and no specific recombinant gelatin is described for use as a plasma extender. Furthermore, there is no description of a method for predicting and controlling the duration of the colloid osmotic effect of the plasma extender.

  A disadvantage of gelatin derivatives that are used commercially is that at least a portion of the administered gelatin passes through the vessel wall and deviates from the circulatory system and cannot contribute to a stable clinical pattern.

  Another disadvantage of the commercially used gelatin derivatives is the fact that the gelatin used is isolated from animal material such as animal bones and skins, in particular from bovine material. The disadvantages of animal materials are the presence of impurities and the lack of reproducibility because the properties of the composition are not clearly defined. This necessitates additional screening to determine whether the derivatization process has reliably obtained a product with the desired properties, and a careful purification step is required. Contamination risk due to gelatin, including the factors involved in the development of Bovine Spongiform Encephalitis (BSE), is an ongoing problem as a further drawback, especially with gelatin isolated from bovine material. This may prohibit the use of gelatin in blood substitute products. At present, at least one product, bovine-derived modified gelatin, is no longer commercially available as a precautionary measure.

  Another disadvantage of gelatin derivatives for commercial use is that when preparing gelatin fragments with the intended size, a completely uniform material is not obtained, which roughly corresponds to the target average molecular weight. Obtained as a heterogeneous mixture of gelatin fragments. Smaller fragments among them impose a negative burden on the renal system without contributing to a stable clinical pattern to be cleared early (unwanted) (high clearance rate) and deviate from the blood circulation.

Another disadvantage of gelatin derivatives currently used as colloidal additives in plasma substitute compositions is that hypersensitivity reactions may occur in subjects. In particular, there is a risk for subjects who have allergies or autoimmune diseases, or subjects whose antibody titer (particularly IgE antibody titer) has increased for some other reason. Severe first aid cases requiring administration of plasma expanders are subjects with shock symptoms, more specifically blood loss shock symptoms caused by massive bleeding, excessive loss of water or insufficient water intake It is for the person. Under such circumstances, there is no time to consider possible risk factors such as whether or not the patient is allergic. If the subject is known to be allergic, antihistamine administration is considered prophylactically. However, in advanced emergency cases, no precautions can be taken. The symptom of direct hypersensitivity that develops with the application of currently used gelatin derivatives is known as anaphylactic shock. This symptom is a life-threatening phenomenon that blood pressure decreases as life cannot be maintained, and is a symptom that actually requires treatment with a plasma expander. Anaphylactic shock symptoms are often fatal because subjects who receive plasma expanders already have acute trauma.
U.S. Pat. No. 2,827,419 International Publication No. 01/34646 Pamphlet

  An object of the present invention is to provide an alternative composition suitable as a plasma substitute containing a plasma expander and having a low clearance rate in the blood circulation system.

  It is also an object of the present invention to provide an alternative composition suitable as a substitute blood plasma, which includes a plasma expander having a better and predictable clearance rate adjusting function than the conventional ones.

  Another object of the present invention is to provide an alternative composition suitable for use as a substitute plasma, comprising a plasma expander having a further reduced sensitivity to proteolysis.

  Furthermore, the object of the present invention includes providing an alternative composition suitable for use as a substitute plasma, which contains a plasma expander that reduces the occurrence of an immune response, particularly anaphylactic shock.

  Surprisingly, it is clear that recombinant gelatin-like proteins having a molecular weight of at least 10,000 Daltons to 25,000 Daltons or up to 50,000 Daltons and having an isoelectric point of less than 8 have a lower clearance rate than conventional gelatin It has become. In the context of the present invention, such gelatin-like proteins are referred to as monomers (monomers) or monomer units. It is possible to prepare gelatin-like proteins composed of repeating monomers or monomer units by recombinant methods. In the context of the present invention, such gelatin-like proteins are referred to as multimers (polymers) or polymers (polymers), in particular dimers (dimers), trimers (trimers) or tetramers (tetramers).

  Surprisingly, it has been found that increasing the number of monomer unit repeats in the polymer gradually decreases the clearance rate. As a result of substituting Glu with Glu or substituting Asn with Asp, as a result of an increase in the charge density of the gelatin-like protein, it is also surprising that a decrease in clearance rate and colloid permeation effect is observed.

  Moreover, surprisingly, recombinant gelatin-like proteins that are substantially free of hydroxyproline do not cause an immune reaction with blood samples containing IgE antibodies.

  Accordingly, the compositions defined in the claims of the present invention are suitable for the purposes of the present invention, and such compositions comprise a physiologically acceptable concentration of saline solution and a protein having a colloid osmotic action, such proteins The colloid osmotic compound is characterized in that it is a recombinant gelatin-like protein having an isoelectric point of less than 8 and a molecular weight of at least 10,000 Daltons to 25,000 Daltons or up to 50,000 Daltons. As a further feature, the present invention also relates to a dimer, trimer or tetramer of a recombinant gelatin-like protein having an isoelectric point of less than 8 and a molecular weight of at least 10,000 Dalton to 25000 Dalton or up to 50,000 Dalton.

  The present invention is a recombinant gelatin-like protein monomer, dimer, trimer, tetramer or other multimeric plasma expander having an isoelectric point of less than 8 and a molecular weight of at least 10,000 Daltons to 25,000 Daltons or up to 50,000 Daltons. It also relates to the usage of.

  The present invention relates to a composition having a monomer, dimer, trimer, tetramer or multimer of a recombinant gelatin-like protein having an isoelectric point of less than 8 as a compound having a colloid osmotic action, wherein the molecular weight of the monomer is at least 10,000 daltons Compositions are provided that are 25,000 daltons or up to 50000 daltons. The molecular weight of the monomer is preferably 10,000 to a maximum of 25000, more preferably 15000 to a maximum of 25000, and even more preferably a maximum molecular weight of 20000.

  By producing a gelatin-like protein by a recombinant method, particularly using a microorganism, it is possible to produce a protein with a constant composition with high reproducibility without risk of prion-related health hazards.

  Example 2 shows that the clearance rate of the composition of the present invention is lower than that of a commercially available composition. It also shows that the repetition rate of the inventive recombinant gelatin monomer in the polymer was repeated many times, resulting in an unexpected decrease in the clearance rate. The part where the colloid is involved in the total osmotic pressure is known as colloid osmotic pressure or oncotic pressure. The composition of the present invention clearly has a hypertonic effect, but in a recombinant gelatin-like molecule that is the same except for different molecular weights, the duration of such a hypertonic effect is extended, which can be used to control the duration of the hypertonic effect. The molecular weight is shown to be available. This superior effect has a very practical connection with the clinical field in which good control of the duration of the colloid penetration effect has been explored in the past.

  In Example 3, in two blood samples in a panel consisting of 60 samples obtained from a subject and the presence of IgE antibodies in the samples, IgE antibodies were specific to these gelatin derivatives for two of the commercial preparations tested. However, in the case of the composition of the present invention, no risk of hypersensitivity reaction was observed for all samples. When a commercial gelatin-based plasma substitute composition is administered as needed to subjects from whom two samples with positive test results are collected, these subjects are likely to develop anaphylactic shock symptoms.

  The primary amino acid sequence of a natural gelatin molecule is basically composed of Gly-Xaa-Yaa triplet repeats, so that about 1/3 of the total number of amino acids is glycine. Usually, gelatin has a large molecular weight, and its molecular weight changes within a range of 10,000 to 300,000 daltons. The molecular weight of the main fraction of natural gelatin molecules is about 90,000 daltons. The average molecular weight exceeds 90,000 daltons.

  Furthermore, a characteristic of gelatin is that it contains an extremely large amount of proline residues. Even more characteristic is that many proline residues are hydroxylated in natural gelatin. Hydroxylation is most prominent at the 4-position, so that the amino acid 4-hydroxyproline, which is not normally present, is present in the gelatin molecule. In the triplet, 4-hydroxyproline is always found in the Yaa position. Very rarely there is a proline residue that is hydroxylated at the 3-position. In contrast to 4-hydroxyproline, 3-hydroxyproline is always found on the carboxyl side of the glycine residue, ie the Xaa position of the triplet. Various enzymes are involved in the formation of 3- or 4-hydroxyproline.

  Based on the known amino acid composition, it is estimated that about 22% of amino acids are proline residues or hydroxyproline residues in mammalian-derived gelatin molecules. However, it has been found that in fish, particularly cold water fish, proline and hydroxyproline are present in lower compositions. As a rule of thumb, approximately 11% of amino acids are proline and approximately 11% are hydroxyproline in mammalian-derived gelatin molecules because they contain approximately the same amount of proline and hydroxyproline residues. Since substantially all hydroxyproline is present at the Yaa position, it is estimated that about 1/3 of all triplets in the gelatin molecule contain hydroxyproline. The presence of a hydroxyproline residue allows the gelatin molecule to take a helix structure in its secondary structure.

  Furthermore, the amino acid other than the above that is present in natural gelatin while hardly existing in other proteins is 5-hydroxylysine. A lysine residue modified in this way is always found at the Yaa position of the triplet.

  The gelatin-like protein used in the present invention is understood as a protein in which at least 5% of the total number of amino acids are proline residues. In view of the object of the present invention, which is defined not as a gelling property but as the absence of an unfavorable three-dimensional spherical domain, the gelatin-like character is ensured under the above proportions. In gelatin-like proteins, it is preferred that at least 10% of the total number of amino acids are proline residues, more preferably at least 15% are proline residues. The lower the proportion of proline in a protein, the more relevant the distribution of proline residues in that protein. Therefore, in a protein in which 5% of the total number of amino acids are proline residues, it is preferable that these proline residues are uniformly distributed. In designing suitable proteins, one skilled in the art can design sequences containing proline residues that do not give rise to globular domains, for example with the aid of a computer model system. As an index for preventing the formation of globular domains, it is preferable that the gelatin-like protein used in the present invention should not contain a stretch of more than 20 amino acids without a proline residue.

  A special feature of gelatin is the presence of Gly-Xaa-Yaa triplets. Such triplets are preferably also present in the gelatin-like protein used in the present invention. However, it is possible to design a protein in which a stretch in which a Gly-Xaa-Yaa triplet or a continuous Gly-Xaa-Yaa triplet is separated by one or more amino acids. In gelatin-like proteins having “interrupted” triplets or continuous stretches of triplets, the gelatin-like characteristics defined above apply. The above definition of gelatin-like protein useful for the present invention is described as a protein comprising a proline residue in at least 15% of the triplet with respect to a protein composed entirely of Gly-Xaa-Yaa triplets. Such gelatin-like proteins preferably do not include stretches longer than 6 triplets in the absence of proline residues. The gelatin-like protein used in the present invention preferably includes a stretch consisting of at least 10, preferably at least 20, more preferably more than 30 consecutive repeats of Gly-Xaa-Yaa triplets.

  In order to maintain a suitable colloid osmotic pressure with the desired clearance rate from the blood circulation when administered to a subject, the molecular weight of the gelatin-like molecule used in the present invention must be at least 10,000 daltons and 20,000 daltons. Is more preferable, and it is more preferable that it exceeds 30000 Dalton. An even more preferred molecular weight is about 30000 Dalton to 120,000 Dalton. Obviously, it is necessary to prepare a multimer of gelatin-like protein as defined in claim 1, at least a dimer, in order to achieve a molecular weight of more than 50,000 daltons. The number of hydroxyproline residues in the gelatin-like molecule used in the present invention is preferably small, meaning that the hydroxyproline residues are less than 10% of the amino acid residues in the polypeptide. The amount of hydroxyproline is limited to prevent gelation of the recombinant gelatin. Gelling is preferably avoided because the concentration of plasma expander applied is limited and it is necessary to warm the plasma extender solution prior to administration.

  The amount of hydroxyproline may be measured by any standard amino acid analysis method. For example, HP AminoQuant Series II, operators handbook, 1990, Hewlett-Packard GmbH, Federal Republic of Germany, Waldbronn Analytical The method described in Division, HP Part No. 01090-90025 is used.

  There are also subjects who are intolerant to commercial blood plasma substitute preparations based on gelatin derivatives, such as those with allergic or autoimmune diseases. As an improvement when confronted with this problem, it was first considered to increase the degree of purification of the gelatin protein. As part of the approach, further optimization of the natural gelatin isolation process, or optimization of the derivatization and subsequent purification steps may be mentioned. Another possibility is to find a material to replace gelatin or another method for producing gelatin. Given the knowledge of biotechnology development and advances in recombinant production of gelatin and collagen seen today, it is considered to aim for the above direction in order to produce a protein with a constant composition with good reproducibility. .

  As mentioned above, gelatin can be fatal for subjects with allergic or autoimmune diseases. When taking the approach of making gelatin recombinantly, given that such gelatin must be less immunogenic in human subjects than currently used bovine derived gelatin, Obviously, manufacturing is employed. It is also clear that no significant changes should be induced in the basic structure of gelatin.

  In one embodiment, the recombinant gelatin-like protein contains less than 10% hydroxyproline. When a hydroxyproline residue is present in natural gelatin, a helix structure is formed in the molecule. By reducing the hydroxyproline residue, the formation of a helix structure in the gelatin-like protein is avoided, and gelation in the protein is avoided even in the low temperature range.

  Information useful in the present invention regarding the immunity and antigenic properties of gelatin-like proteins is not found in the prior art. There are opinions against the use of such proteins in plasma substitute compositions on the basis that the gelatin-like proteins used in the present invention are chemically and structurally different from native gelatins. Surprisingly, however, the gelatin-like protein used in the present invention does not show an immunogenic interaction with blood with an increased IgE antibody titer.

Gelatin-like proteins can be generated de novo from synthetic nucleic acid sequences. This method makes it possible to design a protein in a custom-made manner. The designed synthetic nucleic acid sequence can be expressed in a suitable microorganism by known recombinant techniques.

  Some characteristics of gelatin-like proteins are shown in relation to the design of gelatin-like proteins used in the present invention. For example, by specifying the specific size or specific size range of a gelatin-like protein, the clearance rate in that gelatin-like protein can be “designed in”. It is further advantageous to use this in combination with the characteristics of the renal system that are known for subjects to be administered gelatine-like proteins (e.g., measured with a creatinine clearance pattern). The size of the gelatin-like protein can be designed, for example, by multimerizing a specific monomer (which may be regarded as a block polymer) representing a specific portion of natural human collagen. By gradually increasing the number of monomers in a multimerized complete plasma expander consisting of 1, 2, 3, 4 or more than 5 monomers of gelatin-like protein, a series of well-defined clearance properties for each A plasma expander can be designed. The amino acid (AA) sequence of the monomer has a lower isoelectric point (IEP) of the polypeptide than the human collagen sequence and is less sensitive to any proteolytic activity present in the target single cell production system such as yeast or fungus. Can be selected by selecting an AA domain indicating The size of the gelatin-like protein is more important for colloid osmotic pressure, as described herein. Furthermore, the number of amino acids having isoelectric points and ionizable residue groups can also be adjusted based on the composition of acidic and basic amino acid residues in gelatin-like proteins.

  The isoelectric point of the recombinant gelatin-like protein of the present invention is less than 8. At pH 8, lysine and arginine are positively charged, glutamic acid and aspartic acid are negatively charged, and glutamine and asparagine are neutral. Glutamine and asparagine can be replaced with their acids by point mutations in the expressed sequence or by deamidation of the recombinant structure after expression. Negative charge groups such as aspartic acid residues or glutamic acid residues are preferably randomly distributed throughout the recombinant gelatinous protein. If desired, design-in may be performed by increasing the number of amino acids having a negatively charged residue group as long as antigenicity does not increase.

  What is important to the present invention is that the recombinant gelatin-like protein is selected or designed to have an appropriate isoelectric point, greatly reducing the clearance rate in the blood circulation. By preparing such a multimer of recombinant gelatin-like protein, the above effect can be further improved while maintaining a desired isoelectric point. The isoelectric point is less than 8, preferably less than 7, more preferably less than 6, and even more preferably less than 5. More preferably, the isoelectric point of the gelatin-like protein is at least 3 and more preferably 4. Accordingly, preferred ranges in the present invention are (at least) to (maximum); 3-8, 4-8, 3-7, 4-7, 3-6, 4-6, 3-5, and 4-5 isoelectrics. It is a gelatin-like protein with spots.

  In one example, deamidation, which converts asparagine and / or glutamine to aspartic acid and / or glutamic acid, increases the amount of negative charge groups below pH 8.

  In one example, the composition of the present invention includes a gelatinous protein that inherently exhibits monodispersity. Monodispersity means that the composition and molecular weight are constant. Compositional changes that occur during the production process by recombination are acceptable. A useful definition of monodispersity in the context of molecular weight is that the molecular weight at least 75% of the total amount of gelatinous protein in the composition falls within the range of ± 10% of the selected molecular weight. The molecular weight chosen depends on the desired colloid osmotic pressure and the desired clearance rate from the blood circulation. In another embodiment, the composition of the present invention includes two or more gelatin-like proteins that are all monodisperse but differ in molecular weight. When the molecular weight is different, the clearance pattern in the circulating blood is also different. Based on the above composition, it is possible to adjust the plasma expanding activity of the composition over a long period of time.

  The starting point for the gelatine-like protein used in the present invention may be an isolated gene encoding a naturally occurring gelatin molecule, which is further processed using recombinant techniques. The gelatin-like protein used in the present invention is preferably similar to a human natural amino acid sequence, with the difference that the hydroxyproline residue is less than 10% of the amino acid residue.

  When the point mutation contained in the gelatin-like protein used in the present invention is less than 1%, such protein is similar to the human natural amino acid sequence. However, substitution of asparagine with aspartic acid and glutamine with glutamic acid are not considered point mutations.

  The gelatin-like protein used in the present invention can be prepared by a recombinant technique disclosed in European Patent Application Publication Nos. 0926543, 1014176, and WO01 / 34646. In order to enable the production and purification of gelatin-like proteins suitably used in the composition of the present invention, reference was made to the examples of European Patent Application Nos. 0926543 and 1014176. Thus, gelatin-like proteins can be generated by expressing a nucleic acid sequence encoding such a polypeptide using a suitable microorganism. In this method, fungal cells or yeast cells are preferably used. Suitable host cells are high expression host cells such as Hansenula, Trichoderma, Aspergillus, Penicillium, Neurospora, or Pichia. Fungi and yeast cells are preferred over bacteria because they are less sensitive than bacteria to inappropriate expression of repetitive sequences. Most preferably, the host does not have high levels of proteases that degrade the expressed collagen structure. From this viewpoint, Pichia or Hansenula is exemplified as a very suitable expression system. Methods for using Pichia pastoris as an expression system are disclosed in European Patent Application Nos. 0926543 and 1014176. The microorganism preferably lacks active post-translational processing mechanisms such as hydroxylation of proline and hydroxylation of lysine, among others. The host to be used may not contain a gene that expresses prolyl-4-hydroxylase. It is preferred that lysyl-hydroxylase need not be present in the host. Together with knowledge of host cells and sequences to be expressed, suitable host cells are selected from known industrial enzyme-producing fungal host cells, particularly yeast cells, based on the parameter requirements set forth herein. One skilled in the art can provide suitable host cells for expressing a recombinant gelatin-like protein suitable for the composition.

  By using molecular biotechnology techniques available today, multimers up to tetramers have already been prepared. The invention also encompasses multimers consisting of more than 4 repeats of monomer units, for example 5, 6, 7, 8, 9, 10, and 11 or more repeats.

  When a protein used in the present invention is produced by a recombinant method, particularly when produced by a method of expressing a recombinant gene in yeast, such protein does not contain cysteine or other mercapto amino acids, and 1-4 position (Met− Xay-Xaz-Arg) preferably has no methionine and arginine. This is because such sequences are sensitive to proteolytic degradation by enzymes. In designing or selecting an amino acid sequence according to the present invention, other than the above, a site having a possibility of being susceptible to proteolysis, that is, a specific amino acid stretch is obvious to those skilled in the art. Is not included in the recombinant gelatin-like protein.

  The protein used in the present invention can be partially or wholly produced by, for example, chemical protein synthesis in addition to the DNA expression method. In this case, the protein includes unnatural amino acids. It's okay.

To obtain the composition of the present invention, gelatin-like protein is dissolved in physiological saline having a physiological pH and physiologically acceptable concentration. The physiological saline is a solution in which Na ⁺ ions and Cl ⁻ ions are dissolved in water. Because the plasma substitute composition is very likely to be administered in large doses, care must be taken that the dilution effect does not disrupt the electrolyte balance. In preparing the composition of the present invention, those skilled in the art can use Na ⁺ ions and Cl ^- ions in appropriate concentrations. The practical effective range is 120 to 170 mmol / l for Na ⁺ ions and 90 to 140 mmol / l for Cl ⁻ ions. If desired, one or more components usually contained in blood may be further contained in the composition of the present invention. For example, the composition of the present invention contains one or more components selected from Mg ²⁺ , K ⁺ , Ca ²⁺ , HPO ₄ ²⁻ , H ₂ PO ₄ ⁻ and glucose in a physiologically acceptable concentration. It is. One skilled in the art can determine the physiologically acceptable concentration for each component. Suitably the composition of the invention also comprises a buffering compound, preferably selected from the group consisting of HCO ₃ ⁻ and lactic acid. It is possible for one skilled in the art to determine the appropriate amount of buffering agent to maintain the composition under a physiologically acceptable pH.

  The composition of the present invention is preferably approximately isotonic or isosmotic with the blood of a human subject, and therefore the osmotic pressure of the composition is preferably in the range of 270 to 300 mOsm.

  The purpose of gelatin-like proteins is to maintain adequate colloid osmotic pressure to ensure adequate blood circulation. In one embodiment of the present invention, the fact that the protein used is characterized by non-gelling is advantageous in that it is possible to use a fairly large size polymer that is not rapidly purified from the circulatory system. It is. In order to function effectively as a plasma expander, the molecular weight of the gelatin-like monomer must be at least 10,000 daltons, but should not exceed 50,000. The preferred molecular weight for the monomer is from 10,000 to 25,000, more preferably from 15,000 to 25,000, and even more preferably 20,000.

  In accordance with the present invention, monomer units are multimerized to take advantage of larger gelatinous proteins when deemed desirable in view of the desired colloid osmotic pressure and / or the desired clearance rate from the blood circulation. Is possible. Use of compositions containing high molecular weight gelatin-like proteins without the risk of gelation or the risk of viscosity becoming too high when proline (Pro) hydroxylation is reduced compared to conventional gelatin it can. However, it is considered unsuitable to use gelatin-like proteins with molecular weights greater than 200,000 daltons in the compositions of the present invention. Although it is not preferred that the molecular weight exceed 100,000 daltons, molecular weight has been limited so far because it contains a lot of hydroxyproline and induces gelation.

  The gelatin-like protein of the present invention has an isoelectric point lower than the pH value of human blood (about pH 8). Such gelatin-like proteins preferably have an isoelectric point of at least about 4 and at most about 7.

  Those skilled in the art can use commercially available and free software to design suitable gelatin-like proteins that can be used in the compositions of the present invention. The isoelectric point of a structure having a known amino acid sequence can be calculated by, for example, JaMBW 1.1, a program of L.I.G.Toldo of Molecular Biology Laboratory (Heidelberg).

  In one of the examples relating to the composition and method of use of the present invention, from the number of negatively charged amino acid residues at pH 8 in the recombinant gelatin-like protein, the number of positively charged amino acid residues at pH 8 in the recombinant gelatin-like protein. The value obtained by subtracting is at least 2 and preferably at least 3.

  Many blood plasma proteins have transport functions. A low isoelectric point reduces the chance that gelatin-like proteins interact with these blood plasma proteins, thus reducing the chance that the blood plasma protein function is inhibited.

  The reason why the clearance rate of the gelatin-like protein of the present invention is low can be explained by the barrier property of glycocalyx covering the blood vessel wall. Glycocalix regulates the transport of substances such as lysates and proteins between blood vessels and surrounding tissues. The exact function and mechanism of glycocalyx, which is the reason why such a function is exhibited, has not yet been elucidated. Therefore, it is appropriate to avoid the interaction between glycocalyx and foreign proteins such as gelatin in the plasma extender. Since the gelatin-like protein of the present invention has a low interaction property with glycocalyx, transfer of gelatin-like protein from blood to surrounding tissues is reduced. For example, replacing Gln with Glu or Asn with Asp to increase the total negative charge of a gelatin-like plasma extender at pH 8 reduces the further interaction between the plasma extender and glycocalix. This is a technique for prolonging the half-life in blood vessels of a plasma expander. Further, it is possible to avoid (temporarily) damage of glycocalyx by the gelatin-like protein of the present invention by the repulsive action of the gelatin-like protein on glycocalyx. Such (temporary) damage leads to an increase in circulating blood volume, including plasma expanders, with the undesirable consequence of lowering blood pressure.

  The compositions of the present invention include an amount of gelatinous protein that develops an osmotic pressure comparable to or slightly above that of human serum albumin in the blood. Measurement of the colloid osmotic pressure in the composition is a routine task for those skilled in the art, for example by using a commercially available membrane osmometer with a suitable semi-permeable membrane blocking at 20,000 daltons. . One skilled in the art can correctly determine the amount of gelatin-like protein suitable for the desired osmotic pressure. The amount of gelatin-like protein in the applicable range is usually 2-8% by weight.

  If desired, a pharmacologically active compound can be introduced simultaneously with the surrogate plasma composition of the present invention. For example, it may be advantageous to introduce drugs involved in the blood clotting process simultaneously. Such a composition is particularly useful when applying a plasma expander during surgery or in blood dilution prior to surgery. Accordingly, another embodiment of the composition of the present invention includes pharmacologically active compounds.

  In order to take advantage of the gelatine-like protein's advantage of sustained circulation time in plasma, it is specifically contemplated to covalently bind a pharmacologically active compound to the gelatine-like protein. A further embodiment of the composition of the invention includes a pharmacologically active compound covalently linked to a gelatinous protein.

  Covalently binding pharmacologically active compounds to proteins is a routine matter for the average organic chemist. For example, in order to bind a carboxyl functional group in a drug to an amino group of lysine in a protein, the carboxyl group may be converted to its active ester using DCC or EDC and NHS that react with a free amine.

  As in proteins, lysine residues are a residue option for covalent bonds in other molecules. Therefore, proteins that are substantially free of lysine residues are undesirable. Conversely, lysine residues must be included and preferably the number of lysine residues present is known. When the number is known, it is possible to estimate how many pharmacologically active compounds are bound to the protein, and thus, an appropriate amount of drug can be administered. The de novo design of synthetic nucleic acid sequences now offers the advantageous possibility of introducing specific amounts of lysine residues, thus creating well-defined gelatin-like proteins with pharmacologically active compounds Is possible. A clear correlation is shown between the time that the protein is cleared and the dose of the drug.

  Drugs bound to proteins do not diffuse from the circulating blood into the stroma after administration. This is particularly advantageous for drugs that must act in the blood vessels. Undesirable side effects due to the drug diffusing into the interstitial fluid and spreading into the subject's body are avoided. Agents that have an intravascular activity profile as well as an extravascular activity profile also benefit from the activity being concentrated in the blood vessel.

  Clearance in the liver and kidneys is minimized to ensure more consistent plasma levels of the drug. The half-life of drugs bound to gelatin-like proteins is increased.

  Drugs suitable for intravascular administration and binding to the protein used in the present invention include blood coagulation inhibition, vasodilation, function of red blood cells, platelets and white blood cells, thrombosis, immune response, and blood of messenger molecules such as hormones. Examples are agents that are involved in intermediate levels. Specific examples include blood pressure regulators such as heparin, β-blockers, angiotensin antagonists, and antibiotics.

  The modification of gelatin-like protein used in the composition of the present invention is not limited to binding with a pharmacologically active compound. In order to improve its properties, it is possible to make modifications other than those described above after recombinant production and isolation of gelatin-like proteins. For example, modifications that affect the isoelectric point or solubility or other relevant properties are advantageous. Care should be taken that these modifications do not introduce elements that could elicit an immune or antigenic response.

The human recombinant gelatin-like polypeptides Hu-1, Hu-3, Hu-4 and Hu-deam were prepared by the recombinant method disclosed in European Patent Application Nos. 0926543 and 1014176.

The encoded mature (processed) Hu-1 amino acid sequence is as follows:

Molecular weight: 18.4 kDa, isoelectric point: 5.35

Similarly, Hu-3, which is a trimer of Hu-1, was also produced.

Similarly, Hu-4 which is a tetramer of Hu-1 was also produced.

Similarly, a Hu-deam in which the glutamine residue was substituted with a glutamic acid residue and the asparagine residue was substituted with an aspartic acid residue to reduce the isoelectric point from 8.7 to 4.6 was produced.

  To prevent proteolysis, arginine was replaced with proline at each residue position of 108, 318 and 336.

Preclinical evaluation of gelatin solutions using rats Filling of vasculature is associated with in vivo colloid penetration activity. Colloid osmotic activity can be measured by examining the increase in plasma volume when a predetermined amount of gelatin is administered. In practice, in order to obtain a good measurable effect, for example, 20 ml / kg (about 30% of the blood volume) is extracted in terms of body weight, and the concentration is close to a predetermined concentration (estimated iso-oncotic concentration). It is possible to exchange the same amount with a solution containing gelatin. The in vivo colloid penetration effect by administering a predetermined amount of gelatin solution can be measured by comparing the actual and expected effects on red blood cell count.

  When macromolecules are cleared from the circulatory system, the plasma volume decreases and the red blood cell count or hematocrit value increases. Thus, measuring the change in red blood cell count reveals the duration of the colloid penetration effect. Smaller macromolecules (less than 30 kD depending on charge and shape) are cleared by the kidneys. Excretion from the kidney can be measured by collecting urine and examining its gelatin concentration. If a large amount of gelatin is discharged, gelatin may precipitate in the tubules and blockage of the tubules may occur. This can be observed under an optical microscope.

  Components of the gelatin solution, especially impurities from yeast, can trigger an inflammatory response. As a result, vascular activation and / or neutrophil activation occurs above all.

  Since it is expected that the half-life will be reached in units of time, 4 hours is considered to be sufficient for the initial experiment. That is, since the entire experiment can be performed under anesthesia, blood pressure measurement and blood collection can be facilitated, and rat distress can be minimized.

  Isocolloid osmotic activity is determined without measuring plasma concentration, but for measuring clearance, an assay method for measuring gelatin in plasma and urine is used. Alternatively, labeled gelatin can be used, although there is a drawback that the property may be changed by the label.

Protocol:
Animal data Species: Rat Strain / Gender: Wister HsdCpb: WU, female Procedure Collection of test solution blood: 20 ml / kg for 10 minutes
Injection of gelatin solution: 20 ml / kg (4-6 ml) in 10 minutes
2. Blood samples Blood: 0.2-1.5 ml blood samples were collected from venous cannulas into syringes and quickly transferred to polypropylene vials containing EDTA after 0, 60, 120 and 240 minutes.
3. Experiment period 240 minutes after administration of the test solution, a lethal dose of pentobarbital was administered to terminate the experiment.

Experimental task a) Blood was centrifuged at 10,000 g for 5 minutes using a glass capillary, and hematocrit was measured.
b) The number of red blood cells was counted using an electronic cell counter (model ZF; manufactured by Coulter Electronics).

Calculation The hematocrit (hct) at each time point is calculated from the red blood cell (rbc) count at each time point. From the rbc number at t = 0, the hematocrit at t = 0 is calculated.

The expected (assumed) capacity is calculated as follows:
i) Calculate the expected blood volume (BV) with no fluid movement:
When t = 0 (ml): 65 (ml / kg) × body weight (kg)
When t ≧ 20: (BVt = 0) −removal amount + injection amount
ii) Expected plasma volume (PV) is calculated in ml assuming no fluid movement and the body hematocrit is equal to the hematocrit in the peripheral blood:
When t = 0 (ml): (BVt = 0) × (1-hct t = 0)
When t ≧ 20: (PVt = 0) −removed plasma volume + injection volume
iii) Expected hematocrit is calculated as (BV-PV) / BV.

The real capacity is estimated as follows:
i) Estimate true BV expected at t = 0, then estimate from the ratio of expected and observed hematocrit:
When t = 0 (ml): 65 (ml / kg) × body weight (kg)
When t ≧ 20: BV predicted value × expected hct / observed hct
ii) Estimate from the expected true PV expected at t = 0, and thereafter from the calculated true BV and observed hematocrit:
When t = 0 (ml): (BVt = 0) × (1-hctt = 0)
When t ≧ 20: estimated true BV × (1−hct)

The amount of increase at t = 60 with the infusion test solution was estimated from the difference between the infusion volume and the estimated true and expected plasma volume. :
i) Increase at t = 60: Injection volume— (expected PVt = 60) + (estimated true PVt = 60)
ii) Increase amount per 1 g of colloid (ml / g)

Results and Discussion Increase in Plasma Volume FIG. 1 shows the increase after addition of physiological saline solution and after addition of 5% human serum albumin (HSA) solution. When calculated from the hematocrit value, the physiological saline solution showed a limited effect that lasts only for a short time, whereas HSA shows a colloid penetration effect that lasts very long. The hematocrit value was lower than expected over the entire observation period. When albumin was injected into this model, an increase of 30 ml per 1 g of albumin was observed 1 hour after the injection.

  FIG. 2 shows the increase as an effect of injecting a 4% solution of recombinant gelatin Hu-3 and Hu-4.

  The hypertonic effect is clearly observed in any solution, but the hypertonic effect of Hu-3 disappears after 240 minutes, and the hypertonic effect of Hu-4 lasts for a longer time (up to 6 hours, shown in this experiment). ) Since the only difference between Hu-3 and Hu-4 is the difference in molecular weight, it can be seen that the molecular weight can be used to adjust the duration of the hypertonic effect. Although methods for better controlling the duration of the colloid penetration effect have been sought in the past for clinical use, the remarkable effects described above are closely related to clinical use. As the molecular weight increases from 55.2 kD to 73.6 kD, the hypertonic effect is extended from 4 hours to 6 hours.

  FIG. 3 is a diagram showing the results of measuring the colloid permeation effect of Hu-deam over time. Hu-deam has a completely natural amino acid sequence. In the amino acid sequence, both amino acids Gln and Asn are substituted with both amino acids Glu and Asp. As a result, the isoelectric point (IEP) ) Has decreased from 9.7 to 4.6. This deamidation is similar to the chemical deamidation characteristic of chemical modifications that convert collagen to gelatin. The effect on immunogenicity has been shown to be limited or absent from long-term clinical use of gelifundol and other plasma extenders based on conventional gelatin.

  The deamidation described above is a safe method for reducing the isoelectric point of natural collagen protein and increasing the charge density. FIG. 3 shows that the hypertonic effect lasts over 6 hours, which is longer than for Hu-3. Although the molecular weight of Hu-3 is 55.2 kD and that of Hu-deam is 48 kD, this example shows that the charge density of recombinant gelatin is an important factor to manipulate the hypertonic effect. By increasing the charge density, the duration of the hypertonic effect is extended. Compared to the native sequence, Hu-deam has three more point mutations (Arg → Pro) at each residue position of 108, 318 and 336, reducing the risk of proteolysis. This is because each arginine residue is known to be a certain risk factor that causes proteolytic cleavage (intracellular or extracellular) during the fermentation process.

  FIG. 4 is a diagram showing a colloid permeation effect when gerifundol is added at a concentration of 4 g / 100 ml. Gerifundol has a very short duration of volume effect (FIG. 4). In addition, a small fraction of gerifundol was found in rat urine.

  From the above results, it is clearly shown that the colloid penetration effect can be manipulated using the molecular weight of the recombinant gelatin, and that multimerization of the basic monomer is actually an effective means for enhancing the colloid penetration effect. The colloid penetration effect of Hu-2 (molecular weight 36.8 kD dimer) was shown to be shorter than that of trimer and longer than that of gerifundol (data not shown).

  Furthermore, replacing Gln and Asn with Glu and Asp is considered an effective way to increase charge density. It has been shown that the colloid penetration effect is further enhanced by increasing the charge density.

Radiation allergen adsorption test (RAS test or RAST)
The RAS test is used to reveal the presence of IgE antibodies against a particular allergen or protein. For details of the RAS test, reference was made to Aalberse et al. J. Allergy Clin. Immunol., 1981, vol. 68: 356-364.

The compositions containing gelatin tested were as follows:
Gerofsin®, Gerifundol®, Hu-3 containing composition, Hu-4 containing composition and Hu-deam containing composition Gerofsin® (modified gelatin 40 g / l, Na ⁺ 154 mmol / l, Cl ⁻ 125 mmol / l), and Gerifundol® (modified gelatin 55 g / l, Na ⁺ 145 mmol / l, Cl ⁻ 100 mmol / l, NaEDTA 0.19 g / l, Ca ²⁺ 0.5 mmol / l, HCO ₃ ⁻ 30 mmol / l) was used commercially.

Hu-3, Hu-4 and Hu-deam are described in Example 1. Gelatin-like proteins Hu-3, Hu-4 and Hu in PBS (Na ⁺ 164 mmol / l, Cl ⁻ 140 mmol / l, HPO ₄ ²⁻ 10.9 mmol / l, H ₂ PO ₄ ⁻ 1.8 mmol / l) -Compositions each containing 55 g / l of deam were prepared.

  Serum from subjects known to cause allergies to certain foods was tested. The serum was selected to be known for the presence of IgE antibodies against food, particularly beef, pork and eggs. Subjects with IgE antibodies to the food may also have IgE antibodies to gelatin. In addition, 49 plasma samples obtained from plasmaferese and selected on the basis of the presence of IgE antibodies against known allergies were tested.

  Gelatin derivatives or gelatin-like proteins were bound to CNBr-activated Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) according to standard binding protocols based on manufacturer's instructions (about 1 μg for 1 mg beads). protein). Using a buffer containing human serum albumin, the concentration of beads was 2 mg per ml.

  250 μl of Sepharose beads conjugated to gelatin derivatives or gelatin-like proteins were incubated overnight at room temperature in the presence of 50 μl of serum or plasma sample. These beads were washed 4 times to remove excess serum or plasma and resuspended in 250 μl medium.

The beads are incubated overnight at room temperature in the presence of 50 μl of ¹²⁵ I-labeled anti-human IgE antibody. This labeled anti-IgE antibody is prepared by standard techniques using chloramine T.

The beads are washed 4 times to remove excess ¹²⁵ I-labeled anti-human IgE antibody. The reactivity in the sample is counted (using positive and negative control groups). The presence of reactivity in the sample indicates that IgE was bound to the gelatin derivative or gelatin-like protein in serum or plasma, thus indicating that a hypersensitivity reaction was induced.

  result

  In a control experiment, a sample determined to be positive is pre-incubated in the presence of Gerofcin® and Gerifundol®. No radioactivity was observed in the RAS test performed after the pre-incubation. The immune response is specific for the gelatin derivative used.

It is a figure which shows the relationship between the time passage after physiological saline and human serum albumin (HSA) injection | pouring, and the plasma increase amount. It is a figure which shows the relationship between the time passage after Hu-3 and Hu-4 injection | pouring, and the plasma increase amount. It is a figure which shows the relationship between the time passage after Hu-deam injection | pouring, and the plasma increase amount. It is a figure which shows the relationship between the time passage after gerifundor injection and gelatin plasma concentration.

Claims (15)

In a composition suitable for use as a substitute plasma comprising a physiologically acceptable physiological saline solution and a protein having a colloid osmotic activity, the protein having a colloid osmotic activity has a molecular weight of at least 10,000 daltons and a maximum of 50,000 daltons and A composition characterized in that it is a recombinant gelatinous protein having an isoelectric point of less than 8. A composition comprising a physiologically acceptable physiological saline solution and a protein having a colloid osmotic activity and suitable as a substitute plasma, wherein the protein having the colloid osmotic activity is a dimer, trimer or tetramer of a recombinant gelatin-like protein. A composition characterized in that it has a molecular weight of at least 10,000 daltons up to 50,000 daltons and an isoelectric point of less than 8. 3. Composition according to claim 1 or 2, characterized in that the recombinant gelatinous protein has a molecular weight of at least 15000 daltons up to 25000 daltons. A composition according to any preceding claim, wherein the recombinant gelatinous protein has an isoelectric point of at least 4 up to 7. The number obtained by subtracting the number of positively charged amino acid residues under pH 8 in the recombinant gelatin-like protein from the number of negatively charged amino acid residues under pH 8 in the recombinant gelatin-like protein is at least 2, preferably at least 3. A composition according to any of the preceding claims, characterized in that A composition according to any preceding claim, wherein the recombinant gelatin-like protein is a human gelatin-like protein. Any of the preceding claims, wherein a recombinant gelatinous protein having an isoelectric point of less than 8 is obtained by replacing glutamine with glutamic acid and / or by replacing asparagine with aspartic acid. A composition according to 1. A composition according to any preceding claim, wherein the recombinant gelatin-like protein is a sequence of Hu-1 or Hu-deam. Use of a recombinant gelatinous protein having a molecular weight of at least 10,000 daltons up to 50,000 daltons and an isoelectric point of less than 8 as a plasma extender. Use of a recombinant gelatin-like protein having a molecular weight of at least 10,000 daltons up to 50,000 daltons and an isoelectric point of less than 8 as a plasma dilator or trimer or tetramer. 11. Use according to claim 9 or 10, characterized in that the molecular weight of the recombinant gelatine-like protein is at least 15000 daltons and up to 25000 daltons. 12. Use according to any of claims 9 to 11, characterized in that the isoelectric point of the recombinant gelatinous protein is at least 4 and up to 7. The number obtained by subtracting the number of positively charged amino acid residues under pH 8 in the recombinant gelatin-like protein from the number of negatively charged amino acid residues under pH 8 in the recombinant gelatin-like protein is at least 2, preferably at least 3. The use according to any one of claims 9 to 12. The use according to any one of claims 9 to 13, wherein the recombinant gelatin-like protein is a human gelatin-like protein. The use according to any one of claims 9 to 14, wherein the recombinant gelatin-like protein is a sequence of Hu-1 or Hu-deam.

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