JP2008007448A - Pharmaceutical composition containing amphiphilic peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition transferring the orally or enterally administered bioactive proteins from the intestinal tract to blood, and thereby enabling oral or enteral administration of bioactive proteins so as to enable to provide medicines much improving patient pain and inconveniences. <P>SOLUTION: The pharmaceutical composition transferring the orally or enterally administered bioactive proteins from the intestinal tract to blood is provided, which pharmaceutical composition comprises bioactive proteins and amphiphilic peptides as ingredients. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition for transferring from the intestinal tract into the blood while maintaining the activity of a physiologically active protein.

  So far, several physiologically active substances consisting of peptides and proteins have appeared in the clinical field and have been used as pharmaceuticals and have shown remarkable therapeutic effects. However, the administration method is currently limited to injections in most cases. This is because peptides and proteins are susceptible to degradation by enzymes (proteases) present in the gastrointestinal tract when administered orally, and a small portion of peptides and proteins administered orally because of their low absorption efficiency from the intestinal tract. However, the main reason is that it does not reach the blood. Especially for the latter, since the intestinal tract plays a role as a barrier to prevent substances that are inherently harmful to the living body from entering the body, the intestinal epithelial cell layer essentially has a molecular weight above a certain level such as peptides and proteins. It is considered that it is very difficult to transfer a physiologically active protein into the blood across this barrier.

  Administering drugs by injection is a heavy burden on patients and physicians, especially if treatment is frequent and long-term, and therefore various methods for enabling oral administration of bioactive peptides and proteins are being considered. It has been. For example, attempts have been made to artificially improve intestinal permeability by coexisting surfactants as additives, but using substances that non-specifically increase intestinal permeability is a substance harmful to the living body. The risk of entering the body cannot be excluded.

  In recent years, it has been found that amphiphilic peptides having a specific sequence have a property of permeating the cell membrane. For example, a peptide having “Primary Amphipathicity” in which a hydrophobic region and a hydrophilic region exist on the primary structure, and a hydrophobic surface and a hydrophilic surface when the peptide has a secondary structure such as an α helix. It has been shown that there are peptides that efficiently pass through the cell membrane and migrate into the cell in the peptide with “Secondary Affinity” that produces a surface. Conjugating proteins, DNA, etc. to such cell-permeable peptides It has been reported that the target substance can be taken into the cell from outside the cell (see Non-Patent Documents 1 and 2).

  Although such a cell-permeable peptide-drug conjugate can be used to permeate the intestinal wall into the blood through the intestinal wall, it has been presented as a possibility, but has been demonstrated. Not. This means that translocation of the drug into the blood through the intestinal wall permeates the two barriers, the brush border membrane on the lumen side and the basolateral membrane on the blood vessel side of the epithelial cells that make up the intestinal wall cell layer. This is thought to be due to the need for functions different from the entry into cells. In addition, since various enzymes (proteases) having peptide-degrading activity exist in the intestinal tract, the cell-permeable peptide to be used may be rapidly degraded and lose its function. Since the ease of this enzymatic degradation depends on the sequence of the peptide, even a peptide that exhibits high cell membrane permeability in cell experiments in vitro may not function in the intestinal tract depending on the sequence. Due to such factors, there have been no known examples that have demonstrated the transfer of bioactive proteins with high efficiency from the intestinal tract to the blood using cell-permeable peptides.

As a method other than using a cell-penetrating peptide, a method in which a physiologically active protein is allowed to coexist with a low molecular weight amino acid derivative having a hydrophobic moiety in the molecule as a permeable carrier has been reported (see Patent Document 1). In this method, it is considered that uptake occurs due to a non-specific and passive weak action between the hydrophobic part in the coexisting amino acid derivative and the cell membrane, and it is difficult to transfer a physiologically active protein into the blood with high efficiency. Inevitably, the amount of carriers used is inevitably increased. This leads to concern about undesirable effects caused by administering a large amount of a carrier, which is a modified amino acid derivative that is not a biological component, to the living body. Moreover, it is not desirable from the viewpoint of cost to require a large amount of carriers.
Biopolymers (2004) volume 76, 196-203 Experimental Cell Research (2001) volume 269, 237-244 JP-T 8-509474

  An object of the present invention is to provide a pharmaceutical composition for transferring a physiologically active protein administered orally or enterally into the blood from the intestinal tract.

  In order to overcome the above problems, the present inventors have studied a means for improving the intestinal absorbability of a physiologically active protein having low blood translocation from the intestine under normal conditions. And a composition containing an amphiphilic peptide was found to be effective.

  That is, the present invention has the following configuration.

  A pharmaceutical composition for transferring a physiologically active protein administered orally or enterally into blood from the intestinal tract, comprising a physiologically active protein and an amphiphilic peptide as components.

  According to the present invention, a physiologically active protein can be orally or enterally administered, and a simple and patient-friendly drug treatment can be achieved as compared with conventional administration methods by injection.

  The present invention is a pharmaceutical composition for transferring a physiologically active protein from the intestinal tract into the blood, comprising a physiologically active protein and an amphiphilic peptide as components.

  The amphipathic peptide used in the present invention has a secondary structure in which a peptide having “Primary Amphipathicity” in which a hydrophobic region and a hydrophilic region exist in a primary structure are present and a peptide is α-helix. A peptide having “Secondary Amphipathicity” that produces a hydrophobic surface and a hydrophilic surface when taken is included (Non-patent Document 1).

  The amphipathic peptide used in the present invention is a peptide in which a hydrophobic region and a hydrophilic region exist in a primary structure, for example, a block A-block B or a block B-block A structure, A is an array of 5-10 amino acids in which 80% or more amino acids are composed of hydrophobic amino acids, and Block B is a sequence of 5-15 amino acids in which 60% or more of the amino acids are composed of hydrophilic amino acids. The peptides that are in line are particularly preferred. The amphiphilic peptide used in the present invention is more preferably one having a basic amino acid as a hydrophilic amino acid. An example of a particularly suitable peptide having such properties is a peptide called pVEC (Non-patent Document 2).

  In the present invention, the hydrophobic amino acid represents an amino acid selected from the group consisting of leucine, isoleucine, tryptophan, phenylalanine, valine, and alanine, and the hydrophilic amino acid refers to serine, threonine, aspartic acid, glutamic acid, lysine, It represents an amino acid selected from the group consisting of arginine and histidine. The basic amino acid represents an amino acid selected from the group consisting of lysine, arginine, and histidine.

  As the amino acid constituting the amphiphilic peptide of the present invention, non-natural amino acids such as derivatives obtained by partially modifying the structure of natural amino acids can be used in addition to naturally occurring amino acids. For example, D-form amino acids are effectively used because they are less susceptible to degradation by proteolytic enzymes present in the intestinal tract.

  The amphipathic peptide used in the present invention can be synthesized using a normal peptide synthesis method.

  The amphipathic peptide used in the present invention can also be prepared by introducing and expressing a gene encoding a sequence containing the desired amphipathic peptide in microorganisms such as E. coli, animal cells, and insect cells. is there.

  The amphipathic peptide used in the present invention can be used by isolating a naturally occurring peptide or decomposing it after isolation.

  The amphiphilic peptide used in the present invention can be used as a single peptide or a mixture of two or more peptides.

  More preferably, the amphipathic peptide used in the present invention is Leu-Leu-Ile-Ile-Leu-Arg-Arg-Arg-Arg-Ile-Arg-Lys-Gln-Ala-His-Ala-His-Ser-Lys. It has the amino acid sequence of

  More preferably, the amphipathic peptide used in the present invention is Leu-Leu-Ile-Ile-Leu-Arg-Arg-Arg-Arg-Ile-Arg-Lys-Gln-Ala-His-Ala-His-Ser-Lys. The amino acid sequence consists of a sequence in which 1 or 2 amino acids are deleted, substituted or added.

  If the amount of the amphipathic peptide used in the present invention is too large, it may exhibit a non-specific damaging effect on the intestinal tract, and it is desirable that the dosage be small in terms of cost. Therefore, the mixing ratio of the physiologically active protein and the amphiphilic peptide in the present pharmaceutical composition is desirably 3000 or less as a numerical value of (number of molecules of amphiphilic peptide) / (number of molecules of physiologically active protein). More preferably, it is 1000 or less, More preferably, it is 200 or less.

  Examples of the physiologically active protein used in the present invention include peptide hormones, enzyme proteins, and antibodies.

  Examples of the physiologically active protein used in the present invention include parathyroid hormone (PTH), calcitonin, insulin, angiotensin, glucagon, glucagon-like peptide (GLP-1), gastrin, growth hormone, prolactin (lutein stimulating hormone), gonadotropin. (Gonadotropic hormone), tropotropic hormone, adrenocorticotropic hormone, melanocyte stimulating hormone, vasopressin, oxytocin, protyrelin, luteinizing hormone (LH), corticotropin, somatropin, thyrotropin (thyroid stimulating hormone), somatostatin (growth hormone stimulating factor) ), Hypothalamic hormone (GnRH), G-CSF, erythropoietin, HGF, EGF, VEGF, interferon alpha, interferon beta, interferon gamma, interferon Ikin acids, superoxide dismutase (SOD), can be mentioned urokinase, lysozyme, the vaccine and the like.

  The physiologically active protein used in the present invention is preferably insulin, calcitonin, parathyroid hormone, growth hormone, interferons, interleukins, and G-CSF, and more preferably insulin.

  These physiologically active proteins may be natural proteins or peptides, or derivatives obtained by modifying a part of the sequence. Moreover, you may be the derivative | guide_body which performed chemical modification, such as polyethyleneglycol (PEG) conversion.

  The size of the physiologically active protein used in the present invention is not particularly limited. However, in order to obtain sufficiently high intestinal permeation efficiency, the molecular weight is desirably 30,000 or less, and 10,000 or less. It is further desirable that

  In addition, the isoelectric point of the physiologically active protein used in the present invention is not particularly limited, but the isoelectric point is preferably 7 or less, and more preferably the physiology having an isoelectric point of 5.5 or less. It is desirable to use an active protein. For example, insulin is a protein having a relatively low molecular weight and an isoelectric point of 7 or less that requires frequent administration, and can be particularly preferably used.

  The physiologically active protein referred to in the present invention is transferred from the intestinal tract into the blood when the physiologically active protein is administered into the intestinal tract when an increase in the blood concentration of the physiologically active protein or expression of pharmacological activity is confirmed. means. The blood concentration of the physiologically active protein can be measured by a commonly used method such as an immunological measurement method. The pharmacological activity can be measured using, for example, the enzyme activity of an enzyme, and the ability to change the function of the target cell or the amount of marker substance produced for a substance that acts on a cell receptor. . For example, if it is the pharmacological activity of insulin, it can be measured using the blood glucose concentration of the administered animal as an index. In order for a physiologically active protein administered orally or enterally to exert a sufficient function as a pharmaceutical, the value of AUC (area under the blood concentration-time curve) is used when the same amount of physiologically active substance is administered subcutaneously. It is desirable that a shift of 3% or more is recognized when it is 100%, and it is more desirable that the shift is 10% or more.

  In the pharmaceutical composition of the present invention, preferably, the physiologically active protein and the amphiphilic peptide are not covalently linked.

  The present invention is a pharmaceutical composition for transferring a physiologically active protein from the intestinal tract into the blood, comprising a physiologically active protein and an amphiphilic peptide as components.

  The pharmaceutical composition of the present invention may contain both pharmaceutically acceptable carriers and additives. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran, Sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, Examples include sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.

  The pharmaceutical composition of the present invention can be used in various forms such as a solution, a solid, and a powder. From the viewpoint of stability and ease of handling, the pharmaceutical composition is formed into a solid or powder by a method such as freeze-drying. Form is preferred, preferably a powder or solid formulation.

  The pharmaceutical composition used for oral administration is preferably enteric-coated preparations such as enteric capsules or the like, or enteric-coated oral by a method such as enteric coating after tableting as tablets. Used as a pharmaceutical composition for administration.

  There are no particular restrictions on the specific form of the method for orally or enterally administering the pharmaceutical composition of the present invention to animals (including humans). For example, as an oral preparation, take a dry or solution form as it is, or take it in a capsule with excipients and take it back to water. It can be taken after being dispersed. It can also be administered directly to the rectum using the form of a suppository. The method for administering a physiologically active protein to an animal is preferably administered orally or enterally.

  INDUSTRIAL APPLICABILITY According to the present invention, oral enteral administration of physiologically active proteins that have been used as injections so far is possible, and a drug that greatly improves patient pain and inconvenience can be provided. Improving the pain and inconvenience of hospital visits caused by these injections not only realizes patient-oriented medical care in the medical field, but also fundamentally changes the concept of protein preparations and creates innovative drugs. Leads to.

  Examples are shown below, but the present invention is not limited to these examples.

Example 1
pVEC promotes intestinal absorption of insulin in situ (rat).

<Method>
1. Preparation of Insulin Solution Human Recombinant Insulin (Wako Pure Chemical Industries) 1.923 mg (= 50 IU = 0.33)
μmol) was weighed and dissolved in 50 μl of 0.1M hydrochloric acid. Methyl cellulose (MC)
Phosphate physiological buffer pH 7.4 (PBS) 1.2 ml and 0.1 M NaOH solution 5
Neutralize by adding 0 μl, and add 1.2 ml of MC-added PBS to a total volume of 2.5 m
l. (Insulin concentration 0.769 mg / ml = 20 IU / ml).

2. Preparation of insulin-pVEC peptide mixture
pVEC (synthesized by Sigma Genosys, sequence: Leu-Leu-Ile-Ile-Leu-Arg-Arg-Arg-Ile-Arg-Lys-Gln-Ala-His-Ala-His-Ser-Lys) 0.609 mg (0.25 mmol) was weighed, and 0.5 ml of the insulin solution prepared in 1 above (insulin 0.385 mg = 0.066 μmol) was added and dissolved to obtain a pVEC-insulin mixed solution. For the control (no pVEC), the insulin solution itself was used for administration.

3. Rat intestinal absorption experiment An SD male rat weighing about 200 g fasted for 24 hours was anesthetized by intraperitoneal injection of sodium pentobarbital 50 mg / kg, and then the abdominal cavity was opened along the midline to expose the intestine. A silicon tube was inserted from the 2-3 cm ileum from the ileocecal junction, and a sonde was inserted from the upper portion of about 10 cm, and a suture was passed through the inner 6 cm portion.

Phosphate physiological buffer pH 7.4 (PBS) preheated to 37 ° C from a sonde
After 40 ml was poured at a flow rate of 5 ml / min to discharge the contents, the silicone tube was capped, and 1 ml of PBS preheated to 37 ° C. was administered from the sonde and stored for 30 minutes.
Immediately after administration, the sonde was capped, the intestine was returned to the abdominal cavity, the incision was closed with a clip, and the patient was rested.

After storage, unplug the sonde and silicon tube, and place it in the loop at 37 ° C in advance.
After pouring 20 ml of PBS heated at a flow rate of 5 ml / min to discharge the contents, a 6 cm portion through which a suture thread was passed in advance was ligated to create a loop. In this loop, 2. 0.5 ml of the pVEC-insulin mixed solution prepared in (1) was administered, the intestine was returned to the abdominal cavity, and the incision was closed with a clip to be rested.

Rats in the experiment were fixed in a dorsal position on a hot plate heated to 37 ° C. with a hot water circulation pump to adjust body temperature.

Before administration and 5, 10, 15, 30, 60, 120, 180, 240 minutes after administration, 0.25 ml of blood was collected from the jugular vein, and blood glucose level was measured using Novo Assist Plus.
The remaining blood was separated into plasma by centrifugation, and the insulin concentration was quantified by enzyme immunoassay.

  For rats administered subcutaneously with the same amount of insulin solution (no pVEC), blood was collected and measured in the same manner, and the blood concentration AUC (area under the time curve) value and blood glucose level lowering activity at that time were taken as 100%. Bioavailability (BA) and pharmacological availability (PA) were calculated.

<Result>
Compared with the administration of control insulin alone, co-administration of pVEC resulted in a clear increase in blood insulin concentration and a clear decrease in blood glucose level (FIGS. 1 and 2).

  The bioavailability (BA) calculated from the results is 0.43% when insulin alone is administered, 15.53% when pVEC-insulin mixed solution is administered, and pharmacological availability (PA). Was 0.07% in the case of insulin alone administration, and 7.75% in the case of administration of the pVEC-insulin mixed solution, showing a remarkable increase of 30 times or more and 100 times or more, respectively.

The result of having measured the insulin concentration in the plasma sample which administered the insulin mixed with the pVEC peptide in the rat intestinal loop, and collected blood over time is shown. Insulin solution shows the result in control rats administered with the same amount of insulin alone. The blood glucose level measurement result in each sample of FIG. 1 is shown. Insulin solution shows the result in control rats administered with the same amount of insulin alone.

Claims (10)

  A pharmaceutical composition for transferring a physiologically active protein administered orally or enterally into blood from the intestinal tract, comprising a physiologically active protein and an amphiphilic peptide as components.   The pharmaceutical composition according to claim 1, wherein the physiologically active protein and the amphiphilic peptide are not covalently linked.   The amphiphilic peptide has an amino acid sequence of Leu-Leu-Ile-Ile-Leu-Arg-Arg-Arg-Ile-Arg-Lys-Gln-Ala-His-Ala-His-Ser-Lys. Or the pharmaceutical composition of 2.   Amphipathic peptides are one or two in the amino acid sequence of Leu-Leu-Ile-Ile-Leu-Arg-Arg-Arg-Ile-Arg-Lys-Gln-Ala-His-Ala-His-Ser-Lys. The pharmaceutical composition according to claim 1 or 2, comprising an amino acid sequence deleted, substituted or added.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the physiologically active protein has an isoelectric point of 7 or less.   The pharmaceutical composition according to any one of claims 1 to 5, wherein the physiologically active protein has a molecular weight of 30000 or less.   The pharmaceutical composition according to any one of claims 1 to 6, wherein the physiologically active protein is selected from insulin, calcitonin, parathyroid hormone, growth hormone, interferons, interleukins, and G-CSF.   The pharmaceutical composition according to any one of claims 1 to 7, wherein the physiologically active protein is insulin.   The pharmaceutical composition according to any one of claims 1 to 8, which is a powder or a solid preparation.   A method for administering a physiologically active protein to an animal, wherein the pharmaceutical composition according to any one of claims 1 to 9 is orally or enterally administered.

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