JPH06228199A - Peptide binding body capable of passing through blood brain barrier - Google Patents

Abstract

PURPOSE:To obtain a peptide binding body capable of passing a physiologically active peptide or protein which does not pass through a blood brain barrier by itself through the fluid barrier and uniformly transporting the peptide or the protein into the whole brain. CONSTITUTION:The peptide binding body is obtained by binding a carrier peptide substantially exhibiting no physiological activity by itself and capable of passing through a blood brain barrier to a physiologically active peptide or protein which does not pass through the blood brain barrier by itself. By this binding body, the physiologically active peptide or protein which does not pass through the blood brain barrier by itself can simply be passed through the blood brain barrier without side effects caused by the carrier peptide and is uniformly transported into the whole brain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for delivering a peptide or protein drug into the brain. More specifically, the present invention utilizes a receptor present on brain capillary endothelial cells to carry a physiologically active peptide or protein and a carrier peptide capable of transporting the peptide or protein into the brain by receptor-mediated transcytosis. And the combination with.

[0002]

2. Description of the Related Art Since tissue capillaries such as muscles have pores and intercellular spaces, hydrophilic drugs in blood are rapidly transferred into intercellular fluid of tissues by an intercellular route. But,
Because there are few pores in the brain capillaries and endothelial cells are strongly connected by connective tissue, blood is generally highly lipophilic or is not ionized at physiological pH. The drug inside is difficult to move from the capillaries to the brain. Such a histological filtering function is performed by the Blood Brain Barr.
ier: BBB), which is an obstacle to drug therapy for brain diseases. For example, interleukin 2 (IL
-2) is a cytokine that is produced by T cells and plays an important role in T cell differentiation and proliferation.
Have been shown to proliferate central nervous system oligodendrocytes, astrocytes and their progenitor cells. However, since it is a protein having a molecular weight of 15,400, it cannot pass through the blood-brain barrier, which makes it difficult to apply clinically. Nerve growth factor (NGF) plays an important role as a neurotrophic factor in maintaining survival of cholinergic neurons in the central system, and is expected as a therapeutic drug for dementia such as Alzheimer's disease. However, since it is a protein having a molecular weight of about 13,000, it cannot pass through the blood-brain barrier, which makes it difficult to apply it clinically. Various methods for delivering drugs to the brain such as direct injection and molecular modification of drugs have been developed so far. One method of molecular modification of a drug is an attempt by Boda et al. To increase lipophilicity and increase penetration of intracellular routes. (Japanese Patent Laid-Open No. 63-277662) However, this method has a limit in terms of molecular weight, and is difficult to apply when the physiologically active substance is a high-molecular peptide, particularly a protein.

As another method, a chimeric peptide utilizing endocytosis by in-vivo peptides such as insulin, insulin-like growth factor (IGF) and transferrin, which have receptors on brain capillary endothelial cells, is used. There is an attempt. For example, Pardridge et al. Have reported a chimeric peptide capable of crossing the blood-brain barrier, which is obtained by binding insulin or the like to a nerve drug (Japanese Patent Publication No. 1-500901). But,
Considering the rate of transfer to the brain, a considerably high dose of insulin is required, and serious side effects due to hypoglycemia are concerned. On the other hand, much research has been conducted on the relationship between the amino acid sequence and structure of insulin and the affinity for hormone receptors.

Insulin is a 6000 dalton polypeptide hormone in which two short polypeptide chains, termed the A and B chains, are composed of certain disulfide bonds. The A chain is a 21 amino acid polypeptide chain with internal disulfide bridges, and the B chain is a 30 amino acid polypeptide chain. The A and B chains are linked by two disulfide bridges. In recent years, human insulin produced by genetic engineering has been used, but pig insulin is generally used as an insulin for diabetic patients, and pig insulin differs from human insulin in that Tyr ^{B30 at} the carboxy terminus of the B chain is used. Only the replacement of Ala ^B30 .
In addition, insulin in four cases causing abnormal insulinemia (Phe ^B25 → Leu ^B25 : Chicago, Phe ^B24 → Ser ^B24 :
Los Angeles, Val ^A3 → Leu ^A3 : Wakayama, Tochigi) lacks the ability to bind hormone receptors, so that the amino acids at the C- and B-chain N-termini play an important role in binding to hormone receptors. It is shown. In addition, the carboxy-terminal component of the B chain affects the biological activity of insulin, and the P at the 25th position of the B chain is affected.
It has been found that he (Phe ^B25 ) is involved in the titer of bioactivity. Nakagawa (Nakagawa) et al removal and amidation of the carboxyl group of the new form C-terminal Phe ^B25 of C-terminal pentapeptide sequence of insulin B chain, (B25-30) pentapeptide deficiency - [Phe ^B25
-[Alpha] -Carboxamide] insulin has been obtained as an analog with activity equivalent to that of natural insulin [Journal of Biological Chemistry (J.B.
iol. Chem. 261, pp. 7332-7341 (1
986)]. In addition, by using them, an insulin analog in which Phe ^B25 was replaced with a few other amino acid residues was used to (B26-30) pentapeptide-deficient- [Tyr ^B25- α
-Carboxamido] insulin and His ^B25 analog have about 2.7-3.0 times higher activity than insulin, (B25-30) hexapeptide deficiency, (B24-
It has been shown that the 30) heptapeptide deficient and (B23-30) octapeptide deficient analogs have little insulin activity. In addition, Pullen (Pull
en) et al., from the study of crystal analysis structure, the important points for expression of biological activity are three residues of A chain (Gly ¹ , Tyr ¹⁹ , Asn ²¹ ),
Five residues of B chain (Val ¹² , Tyr ¹⁶ , Phe ²⁴ , Phe ²⁵ , Tyr
^It has been pointed out that the lipophilic receptor binding site containing the amino acid is stabilized in terms of molecular geometry [Nature 259, 369-373 (197).
6)]. These conventional studies have reported a lot of information on insulin analogues showing an affinity for hormone receptors showing hypoglycemic action, but no report on receptors on brain capillaries.

[0005]

A method of transporting a physiologically active peptide or protein through the blood-brain barrier and transporting it into the brain is to uniformly transport the peptide or protein throughout the brain and minimize side effects. Is desired. However, a transport form of a physiologically active peptide or protein that can sufficiently meet this requirement has not been established.

[0006]

Based on such a technical background, the inventors of the present invention are carriers that are permeable to the blood-brain barrier and exhibit no side effects in transporting a physiologically active peptide or protein into the brain. As a result of diligent efforts to find peptides, an insulin fragment showing an affinity for the insulin receptor on brain capillary endothelial cells and having less hypoglycemic effect than insulin penetrates the blood-brain barrier by receptor-mediated transcytosis However, they discovered the fact that they could be carriers for bioactive peptides and proteins. Based on this finding, it is possible to utilize the physiological process of transcytosis mediated by receptors in the blood-brain barrier to transport peptides or proteins that do not themselves cross the blood-brain barrier bound to their carrier peptides. The present invention has been completed by further researching a carrier peptide that can be produced and can prevent harmful side effects due to the activity of the carrier peptide itself and its transport form.

That is, the present invention comprises: 1) combining a physiologically active peptide or protein that does not itself cross the blood-brain barrier with a carrier peptide that does not exhibit physiological activity itself and that can cross the blood-brain barrier. It is a peptide conjugate that can cross the blood-brain barrier. More specifically, 2) the carrier peptide has a molecular weight of about 4,000.
The conjugate according to 1 above, which is a peptide of 0 to 100,000, 3) The conjugate according to 1 above, wherein the carrier peptide is an inactive fragment of a peptide in vivo that can cross the blood-brain barrier by receptor-mediated transcytosis. 4) In vivo peptide is insulin, transferrin, insulin-like growth factor I or II
The conjugate according to 5), wherein the carrier peptide is a peptide chain having 14 to 21 amino acid residues from the N-terminus of the A chain of insulin (SEQ ID NO: 1) and a peptide having 16 to 22 amino acid residues from the N-terminus of the B chain. The conjugate according to 1 above, which is an insulin fragment consisting of a chain (SEQ ID NO: 2), and 6) the insulin fragment

[Chemical 2] The conjugate according to the above 5, which is a fragment represented by:
7) The conjugate according to 5 above, wherein the insulin fragment is an insulin fragment lacking (B23-30) octapeptide, and 8) the conjugate according to 1) above, wherein the physiologically active peptide or protein has a molecular weight of about 200 to 120,000. 9) The conjugate according to 1 above, wherein the physiologically active peptide or protein has a molecular weight of about 400 to 80,000, and 10) the conjugate according to 1 above, wherein the physiologically active peptide or protein has a molecular weight of about 500 to 60,000. 1
1) The conjugate according to 1 above, wherein the physiologically active peptide or protein is a neurotrophic factor, 12) The conjugate according to 1) above, wherein the physiologically active peptide or protein is a peptide or protein belonging to the neurotrophin family, 13) a physiological activity The conjugate according to 1 above, wherein the peptide or protein is nerve growth factor (NGF), 14) the conjugate according to 1 above, wherein the physiologically active peptide is a neuropeptide, 15) a peptide or protein belonging to the neurotrophin family and an insulin fragment 16. The conjugate according to 1, wherein 16) nerve growth factor (NGF) and (B23-30)
The conjugate according to the above 1, which is formed by binding an octapeptide-deleted insulin fragment.

The conjugates of the present invention are useful for transporting various bioactive peptides or proteins to the brain. In particular, it is generally optimal for the delivery of high molecular weight proteinaceous drugs that are poorly transported across the blood-brain barrier. Examples of these peptide or protein drugs include various neurotrophic factors and neuropeptides which are useful as drugs for treating brain diseases which cannot pass through the blood-brain barrier by themselves. Neurotrophins include neurotrophin family such as nerve growth factor (NGF), brain-derived neurot
rophine factor (BDNF), neurotropin 3
(NT-3), neurotrophin 4 (NT-4), neurotrophin 5 (NT-5) and ciliaryneurot
rophic factor (CNTF), activin, or basic fibroblast growth factor (bFGF) that also works in the central system,
Growth factors such as acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF), or directly or indirectly acting on the nervous system, involved in proliferation, differentiation and activation of glial cells, neurotrophic factor Cytokines having action, for example, interferon α, interferon β, interferon γ, interleukin 1 (IL-
1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-)
4), interleukin 5 (IL-5), interleukin 6 (IL-6), TNF, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), platelet-derived growth factor (PDGF), and hormones or neurotransmitters that act in the brain, such as somatostatin, oxytocin, vasopressin, galanin, VIP, adrenocorticotropic hormone (ACTH),
Cholestokinin (CCK), substance-P, bombedin, motilin, glicentin, glucagon, glucagon-like peptide (GLP-1), peptide YY (PY
Y), neuropeptide Y (NPY), pancreatic polypeptide (P
P), neurokinin A, neurokinin B, endorphin, enkephalin, neurotensin, neuromedin K, neuromedin L, calcitonin-related peptide (CGRP), endothelin, cardiac natriuretic peptide (ANP), brain natriuretic peptide (B).
NP), C-type natriuretic peptide (CNP),
Pituitary adenylate cyclase stimulating peptide (PA
CAP) and other neuropeptides and their active derivatives. Enzymes such as horse radish peroxidase (HRP) can also be used as the physiologically active peptide or protein of the present invention.

As described above, in the present invention, the physiologically active peptide or protein targeted for transport is generally a polypeptide having a molecular weight of 200 daltons or more, and is hydrophilic and cannot pass through the blood-brain barrier by itself. As long as it is a natural type derived from an animal or plant, it may be one obtained by a known peptide synthesis method or genetic engineering method. Further, examples of the above-mentioned active derivative include optical isomers, active fragments and muteins. Here, the mutein is a mutated amino acid sequence of the original protein due to addition of amino acids, deletion of constituent amino acids, substitution with other amino acids, and the like, which has physiological activity of the original protein. Some of them are listed. The mutein may be a combination of two or more mutations of addition, deletion and substitution. Examples of the addition of amino acids include muteins to which at least one amino acid is added. The additional amino acids in the muteins include methionine and signal peptide derived from the initiation codon used when expressing the peptide. Make it not exist. As the number of additional amino acids,
At least one, but any number may be used as long as the original physiological activity is not lost. The constituent amino acid deficiency includes a mutein in which at least one constituent amino acid is deleted, and the number of the constituent amino acids lacking may be any number as long as it does not lose its own physiological activity. .

Examples of substitution with other amino acids include muteins in which at least one constituent amino acid is replaced with another amino acid, and the number of constituent amino acids before the replacement is at least one. Any number may be used as long as it does not lose its original physiological activity. In the present specification, “passing through the blood-brain barrier” means that when a physiologically active peptide or protein is administered to the blood-brain barrier within a range of the usual permissible dose, the original physiological activity of the peptide or protein causes It means that it exhibits permeability to the administration subject to such an extent that physiological activity, for example, a therapeutic effect on brain disease can be produced.
Therefore, a physiologically active peptide or protein having extremely low permeability to the blood-brain barrier and its derivative are also included in the category of the physiologically active peptide or protein which does not cross the blood-brain barrier in the present invention. The molecular weight of a physiologically active peptide or protein such as a drug for treating brain disease which can be transported by the conjugate of the present invention is slightly different depending on its three-dimensional shape, size, stability to enzyme, water solubility, etc. There is no limitation, but generally includes proteins of about 200 daltons or more and up to about 120,000 daltons. Above all, it is a drug having a molecular weight of about 400-80,000 daltons that more effectively exerts the transporting ability of the conjugate of the present invention, and more preferably about 500-60,0.
The one of 00 Dalton is mentioned.

Examples of the carrier peptide of the present invention include peptides that have substantially no physiological activity themselves and preferably have an affinity on brain capillary endothelial cells. Particularly preferred is a peptide having a receptor on the cell, but a conjugate formed by binding with a physiologically active peptide or protein that does not pass through the blood-brain barrier itself passes through the blood-brain barrier. Any peptide may be used as long as it is a transmissive peptide. Such a carrier peptide usually has a molecular weight of about 4,000.
To 100,000 peptides are used. Also,
As used herein, the term "peptide having substantially no physiological activity per se" means that a physiologically active phenomenon is not caused in a living body at an ordinary dose, or the physiologically activated phenomenon is not a physiologically normal phenomenon. Peptides within the range of variation are indicated. Therefore, the carrier peptide of the present invention may have activities that are not directly involved in physiological activities such as membrane permeability and receptor affinity, and in the present invention, in particular, receptor-mediated transcytosis on brain capillary endothelial cells. It is preferable to have a tosis activity. From the viewpoint of antigenicity, safety, etc., it is preferably a mammalian in vivo peptide, particularly preferably an in vivo peptide of an administration target animal, and particularly preferably an in vivo peptide capable of passing through the blood-brain barrier. An inactive fragment or a derivative in which the original physiological activity is diminished or eliminated while maintaining the barrier crossing property is preferably used. Examples of the carrier peptide of the present invention include inactive peptide fragments such as insulin, transferrin, insulin-like growth factor I (IGF-1), insulin-like growth factor II (IGF-II), and amino acids of these peptides. Examples include peptides having affinity on brain capillary endothelial cells obtained by combining a part of the sequences. These peptide fragments can be obtained by various methods, and can be obtained, for example, by degrading insulin or the like with a protease such as trypsin, chymotrypsin, pepsin, papain or V8 protease.

In particular, when an insulin fragment is used as the carrier peptide, the preferred range of the insulin fragment is a peptide chain having 14 to 21 amino acid residues (SEQ ID NO: 1) and B from the N terminus of the A chain.
An example is a fragment consisting of a peptide chain having 16 to 22 amino acid residues (SEQ ID NO: 2) from the N-terminal of the chain. Moreover, in order to maintain the original affinity for brain capillary endothelial cells in these fragments, a disulfide bridge involved in the three-dimensional structure, for example, an intramolecular bridge between amino acid residues Nos. 6 and 11 of the A chain. It is preferable that the crosslink between A chain and B chain is maintained at the same position as that of the naturally occurring whole molecule protein. Among them, the (B23-30) octapeptide-deficient insulin fragment [Fig. 6] obtained in Example 2 described below has a similar affinity to insulin for receptors on brain capillary endothelial cells. This is unexpected from the conventional knowledge of the affinity of insulin analogues for hormone receptors and can be cited as an example of the preferred carrier peptide in the present invention. These carrier peptides can also be obtained by known peptide synthesis techniques. For example, one example thereof is a solid phase synthesis method using a polystyrene resin as a carrier. In this method, the amino acid residue at the carboxy terminus is covalently bound to the resin carrier, the α-amino protecting group is removed, and the protected amino acid is sequentially repeated to extend the peptide toward the amino terminus to obtain the desired amino acid sequence. The principle is to obtain the protected peptide resin. When the target carrier peptide is a peptide consisting of two or more peptide chains, for example, in the case of an insulin fragment (double chain), the two peptides obtained by separately synthesizing each of the two chains beforehand are obtained. By reacting the chains under weak alkalinity, a disulfide bond can be easily formed, and the target carrier peptide crude product can be obtained. The obtained crude carrier peptide product can be purified by a known means for purifying peptides or proteins. Examples include purification methods such as gel filtration, ion exchange chromatography, hydrophobic chromatography, partition adsorption chromatography and high performance liquid chromatography.

It can also be obtained by a genetic engineering method using microorganisms such as bacteria and fungi and biological cells such as hamster egg cells as hosts. The conjugate of the present invention is
It can be obtained by binding a physiologically active peptide or protein to a carrier peptide, and the binding method may be a known chemical or genetic engineering method. Specifically, in the case of a chemical bond, 1-ethyl-3- (3-dimethylaminopropyl) that forms a peptide bond
A cross-linking agent that can react with any of two peptide molecules (that is, a physiologically active peptide or protein and a carrier peptide) such as carbodiimide (EDAC, WSC) is used. Among them, a method applying peptide thiolation characterized by treating with a reagent such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) or cystamine which forms a disulfide bond between two peptides. Is preferred. As for other known binding agents, as long as two peptide molecules can be bound without deteriorating both the blood-brain barrier permeability of the carrier peptide and the physiological activity of the physiologically active peptide or protein by denaturation, Any binder may be used. More specifically, the following cross-linking agent is selected from the reactivity with the functional group and the length of the spacer. Glutaraldehyde, 4,4'-diisothiocyanostilbene-
2,2'-disulphonic acid (DIDS), N- (E-maleimidocaproyloxy) succinimide (EMC
S), N- (gamma-maleimidobutyloxy) succinimide (GMBS), dithiobis (succinimidylpropionate) (DSP), and 4,4′-dithiobisphenylazide (DTBPA) as a photoactivating reagent. There is.

Further, these bound products can also be synthesized by an ordinary liquid phase method or solid phase method by an amide bond or a disulfide bond using a reducing agent. further,
These bioactive peptide conjugates may be directly expressed by known genetic engineering techniques using microorganisms such as bacteria and fungi and biological cells such as hamster egg cells. That is, for example, a cDNA obtained by obtaining a cDNA corresponding to the carrier peptide of the present invention from a known cDNA encoding insulin, and directly binding a cDNA encoding a physiologically active peptide or protein to be transported to the cDNA.
Can be obtained by transforming a suitable host cell with the vector containing this. In the conjugate of the present invention, the binding ratio of each of the physiologically active peptide or protein constituting the carrier and the carrier peptide can be selected according to the delivery ability of the carrier, the activity of the drug and the resilience to the active substance when the conjugate is used. it can. The binding molar ratio of the carrier to the physiologically active peptide or protein as a drug is preferably 4: 1 to 1: 5, more preferably 2: 1 to 1: 3.

In the present invention, the ratio of the physiologically active peptide or protein used as a drug to the carrier peptide is a combination of both peptides used, that is, a functional group involved in the binding reaction of the protein in each peptide chain, such as the N-terminal,
C-terminal, basic amino acids (arginine, lysine, histidine, etc.) acidic amino acids (glutamic acid, aspartic acid, etc.) and cysteine residues can be changed according to the selection of the type and number, and the type and concentration of the reagent, It can be controlled by controlling reaction conditions such as reaction time. The thus obtained conjugate of the present invention is a warm-blooded animal, particularly a mammal (cow, horse, dog, cat, human etc.) for the purpose of prevention and treatment of various brain diseases such as brain tumor, cerebrovascular disorder and Alzheimer's disease. ), A single or several kinds thereof can be formulated and administered in combination, and other pharmacologically acceptable drugs can be further compounded and safely administered. As a method for administering the conjugate of the present invention, parenteral administration is preferable, and for example, it can be administered as an ordinary injection in a blood vessel, intramuscularly, subcutaneously, etc., and particularly preferably intravenously or intraarterially. In the preparation of the injectable composition of the conjugate of the present invention, a preservative, a stabilizer, an isotonicity agent and the like usually used for injectable agents may be added. For example, in the case of preparing an aqueous solution, an oil solution, and a suspension injection, distilled water for injection, an aqueous solvent (physiological saline solution, Ringer's solution), an oil solvent (eg, sesame oil, olive oil), or the like, or if desired. Dissolution aid (eg Tw
een-80, arginine), buffer (eg sodium phosphate, sodium citrate, sodium acetate), isotonicity agent (eg glucose, glycerin, sorbitol), stabilizer (eg human serum albumin, polyethylene glycol) ), A preservative (eg, benzyl alcohol, phenol), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride), and the like.

Further, for example, in order to prepare a solid injectable preparation which is dissolved or dispersed at the time of use, a solution and a dispersion medium (eg,
Distilled water, physiological saline, glucose solution), excipients (eg, carboxymethyl cellulose (CMC), mannitol,
Sodium alginate), preservative (eg, benzyl alcohol, benzalkonium chloride, phenol), soothing agent (eg, glucose, calcium gluconate, procaine hydrochloride), etc. are mixed and manufactured into a solid injection preparation by a conventional method. can do. Examples of preparations other than injections include preparations for mucosal administration in the nose, oral cavity, rectum, vagina, uterus, sustained release preparations such as microcapsules, and controlled release preparations such as implants. In formulating these, monosaccharides such as glucose, amino acids, various salts, human serum albumin and the like may be added, and other usual isotonic agents, pH adjusting agents, soothing agents, antiseptics, etc. And absorption enhancers known per se (eg, citric acid, α-
Cyclodextrin, mandelic acid, sodium salicylate, bile acid, pooxyethylene (9) lauryl ether, taurodihydrofusidic acid) and the like can be added to prepare stable and effective conjugate formulations. The conjugate of the present invention is, for example, a human trophic factor such as human NGF or B.
For the purpose of administration of DNF, NT, FGF, etc., a relatively low dose and a sufficiently useful effect can be expected to suppress or treat the progression of Alzheimer's disease. Moreover, various enkephalins, somatostatin, substance-P, VIP, etc., which are neuropeptides that act in the brain, can be efficiently delivered to the brain according to the present invention, and thus, a similar reduction in dose and reduction in side effects in the periphery can be expected. . The human dose of the conjugate of the present invention varies depending on the physiologically active peptide or protein to be used, the subject to be administered, the symptoms, the mode of administration, etc., but may be any amount as long as it exhibits the required pharmacological effect, and is usually 10 μg to
2 g, more preferably 20 μg to 1 g, is parenterally administered in 1 to 3 times daily.

The conjugate of the present invention is not only a receptor on brain capillary endothelial cells but also receptors such as insulin, transferrin, insulin-like growth factor I or II present on various organs such as liver and kidney or cancer cells. Can be used to detect For example, a conjugate of a carrier peptide and a physiologically active peptide or protein that serves as a marker for an enzyme (horse radish peroxidase, etc.) can be used for this purpose, and an antibody against the physiologically active peptide was used. It can also be used for detection in combination with an immunoassay. Thus, the conjugate of the present invention, for example, the conjugate of an inactive fragment of insulin and a marker peptide, is useful for elucidating insulin receptor deficiency, such as hepatocytes, lymphocytes, adipocytes, and stars. It can be usefully used to elucidate the action mechanism of insulin on various cells such as glial cells, nerve cells, and glial cells. Further, for example, in congenital or acquired obesity, it can be used to detect whether it is caused by insulin receptor deficiency or whether it is responsive to insulin in cancer cells such as liver cancer and lung cancer. it can.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IU
It is based on the abbreviations by B Commision on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: Deoxyribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine Gly: Glycine (G) Ala: Alanine (A) Val: Valine (V) Leu: Leucine (L) Ile: Isoleucine (I) Ser: Serine ( S) Thr: Threonine (T) Cys: Cysteine (C) 1 / 2Cys: Half cystine Met: Methionine (M) Glu: Glutamic acid (E) Asp: Aspartic acid (D) Lys: Lysine (K) Arg: Arginine (R) ) His: Histidine (H) Phe: Phenylalanine (F) Tyr: Tyrosine (Y) Trp: Tryptophan (W) Pro: Proline (P) Asn: Asparagine (N) Gln: Glutamine (Q)

[0019]

[Examples] Formulation examples, reference examples and examples are shown below,
The present invention will be described in more detail, but these are merely examples and do not limit the present invention in any way. Reference Example 1: Blood-brain barrier permeability test ¹²⁵ I-labeled specimen-carrier peptide conjugate was dissolved in physiological saline and administered to ICR mice.
Uptake of ¹²⁵ I-labeled analyte is measured. At each time
¹²⁵ I ¹²⁵ I, prepared similarly uptake labeled analyte brain
The in vivo transport capacity of the carrier peptide is evaluated by comparing with the case where the labeled sample is administered alone.

Example 1: Preparation method and identification of F001 100 mg of porcine-insulin (Shimizu Pharmaceutical Co., Ltd.) dissolved in 2.5 ml of 10 mM hydrochloric acid was dissolved in 0.1 M phosphate buffer (pH 7.0) 50.
The mixture was diluted with ml, trypsin (Sigma) (5 mg) was added thereto, the mixture was left standing at room temperature for 3 days, and the whole was freeze-dried. The obtained decomposition mixture was dissolved in 1N acetic acid, the insoluble matter was removed with a 0.45 μm filter, and then the reversed phase preparative high-performance liquid was eluted with a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid (TFA). Chromatography (HPL
C) (Column: TSK gel ODS-120T, 2.15
X30 cm; flow rate 0.7 ml / min; acetonitrile concentration 10% for the first 3 minutes, then elution with a concentration gradient from 10% to 60% over 25 minutes) to collect the main peak and freeze-dry. Concentrated to give a white powder. The obtained insulin fragment was named F001.
F001 was a single peak ([FIG. 1] and [FIG. 2]) separated from the insulin peak by reverse phase analytical HPLC, and the yield was 6.5%. F001 was determined to have a structural formula as shown in FIG. 3 from the result of analysis by an amino acid sequencer.

Example 2: Preparation and identification of F007 Porcine-insulin 300 dissolved in 2.5 ml of 10 mM hydrochloric acid.
mg was diluted with 150 ml of 0.1 M N-ethylmorpholine (pH 8.0) containing 2 mM EDTA, and added to this to 20 mM.
45 mg of trypsin dissolved in 150 ml of 0.1 MN-ethylmorpholine containing calcium chloride was added, and the temperature was 37 ° C.
And incubated for 90 minutes. 1N acetic acid, 10 ml
Was added to stop the reaction, and the whole amount was freeze-dried. The obtained decomposition mixture was dissolved in 1N acetic acid, the insoluble matter was removed by a 0.45 μm filter, and then the reverse phase preparative H was eluted with a gradient of acetonitrile / water containing 0.1% TFA.
PLC (column: TSK gel ODS-120T, 2.1
5 × 30 cm; flow rate 0.7 ml / min; acetonitrile concentration 10% for the first 3 minutes, then 10% over 25 minutes
To 60% by elution with a concentration gradient), the main peak was collected and concentrated by freeze-drying to obtain a white powder.
The obtained insulin fragment was named F007. F007 was a single peak (FIG. 4 and FIG. 5) separated from the insulin peak by reverse phase analytical HPLC, and the yield was 29.5%. F007 was determined to have a structural formula as shown in FIG. 6 from the results of amino acid analysis [Table 1] and the structural formula of insulin after hydrolysis with 6N hydrochloric acid.

[0022]

[Table 1] Results of amino acid analysis of F007 Insulin F007 ───────────────────────────── Amino acid analysis results Theoretical values Analysis results Theoretical values ── ────────────────────────── Asp 2.9 3 2.9 3 Glu 6.7 7 6.3 7. 7 Ser 2.8 3 3 3 Gly 4.2 4 3.5 3 His 1.9 2 1.9 2 Arg 1.1 1 1 1 1 Thr 1.9 2 1.1 1 Ala 2.1 2 1.2 1.2 1 Pro 1.1 1 0 .2 0 Tyr 4.1 4 3.3 3 Val 3.5 4 3.6 4 Cys 2.5 6 2.6 6 Ile 1.6 2 1.7 2 Leu 6 6 6 6 Phe 3.1 3 1 .5 1 Lys 1.1 1 0.2 0 ─────────────────────────────

Example 3: Receptor Affinity of Insulin Fragment (1) "Characteristics of the Large Neutral Amin System on Bovine Capillary Endothelial Cells" by Ronald T. Borchart et al.
o Acid Transport System of Bovine Brain Microvesse
l endotherial cell Monolayers) "[Journal of Neurochemistr
y) Vol. 47, p. 484-488 (1986)], bovine brain capillary endothelial cells were separated, a monolayer was prepared and the affinity for the receptor on the endothelial cells was measured. That is, ¹²⁵ I was added to the cultured cells reaching the packed structure.
-Insulin coexistence was added with the insulin fragments (F001, F007) obtained in Examples 1 and 2 and unlabeled insulin, respectively, and the affinity for the insulin receptor present on capillary endothelial cells was determined for ¹²⁵ I-insulin. It was examined in a competitive inhibition experiment. As shown in [Table 2], both F001 and F007 show affinity,
F007 showed a similar affinity to all insulin molecules. As previous studies have shown, affinity for cultured brain capillaries is a reliable indicator of brain capillary permeability in vivo, and this example is directed to the brain by transcytosis via the insulin receptor. Was sufficiently suggested.

[0024]

[Table 2] Comparison of affinity of insulin with F001 and F007 for receptors on brain capillary endothelial cells Binding rate of ¹²⁵ I-insulin when unlabeled compound was added (%) ──────────── ───────────────────── Additive concentration (μM) Insulin F001 F007 ───────────────────── ─────────── 1.6 X 10 ² 13.6 ± 0.2 1.0 X 10 ² 61.6 ± 1.2 11.8 ± 2.5 1.0 X 10 ² 23.0 ± 6.3 78.5 ± 3.8 18.1 ± 2.6 1.0 X 10 24.2 ± 1.5 93.2 ± 0.4 33.5 ± 1.7 1.0 X 10 ^-1 30.2 ± 3.9 55.7 ± 6.6 1.0 X 10 ^-2 44.6 ± 0.9 73.7 ± 8.8 1.0 X 10 ^-3 73.7 ± 8.3 90.3 ± 2.8 1.0 X 10 ^-4 85.7 ± 8.5 94.7 ± 10.8 ─── ─────────────────────────────

Example 4: Synthesis of F007 A chain and B chain of F007 were synthesized by a solid phase synthesis method using polystyrene resin as a carrier. A t-butoxycarbonyl (tBOC) group was used for the N (α) -amino group, and a benzyl type protecting group was used for the functional group of the amino acid side chain. In addition, cysteine residues involved in disulfide bonds in the A and B chains of F007 (A chain 7-position and 20-position
Position, B chain 7-position and 19-position), acetamidomethyl (A
cm) group was used as a protecting group. Starting from a polystyrene resin having a C-terminal amino acid bonded as a starting material, the tBOC group was sequentially removed with 0.5% methanesulfonic acid, and then washed with 2% pyridine, and then the tBOC-protected amino acid was benzotriazol-1-yl-yl-. Condensation was achieved by activation with oxy-tris (dimethylamino) -phosphonium hexafluorophosphate. The protected peptide resin for which the entire condensation reaction was completed was dried under reduced pressure. The dried protected peptide resin was stirred in hydrogen fluoride for 2 hours under ice cooling in the presence of a suitable cation scavenger to remove the peptide from the resin and remove the protecting group. Hydrogen fluoride was distilled off under reduced pressure, and ether was added to the residue to precipitate and precipitate the peptide. Further, it was washed three times with ether to remove hydrogen fluoride and scavengers sufficiently. Next, the obtained crude peptide was extracted with 1N acetic acid, the insoluble matter and the resin were filtered off with a glass filter, and the filtrate was freeze-dried. A small amount of 1N was added to the dried product.
It was dissolved in acetic acid and added to a Sephadex G-25 (Pharmacia) column (2.7 × 90 cm) to give 280 n.
While monitoring the absorbance at m or 254 nm, elution was performed with 1N acetic acid, and the fractions containing the peptide of interest were collected and lyophilized.

For the A-chain peptide containing an unprotected cysteine residue, dilute aqueous ammonia (pH 7.6) was added in an amount 1000 times that of peptide 1, and the mixture was allowed to stand at room temperature for 48 hours.
Disulfide bonds were formed and freeze-dried. The obtained dried product of A chain and B chain was dissolved in 1N acetic acid, and then TSK g
el ODS-120T (2.15X30cm) Acetonitrile containing 0.1% TFA by reverse phase preparative HPLC-
It was purified by gradient elution with an aqueous system. The target peak was freeze-dried to obtain a white powder. The purity of these peptides was confirmed by reverse phase HPLC, and it was confirmed from the results of amino acid analysis after hydrolysis with 6N hydrochloric acid that the peptides were the target peptides.
Next, the Acm groups of the A chain and the B chain were deprotected by iodooxidation, and a Sephadex G-25 column (2.7x9 was used.
0 cm) and eluted with 1N acetic acid and lyophilized. The A chain and B chain corresponding to F007 were allowed to stand at room temperature for 24 hours in diluted ammonia water (pH 7.6) to form a disulfide bond between the A chain and B chain. After confirming the progress of the reaction by reverse phase analytical HPLC, TS
Kgel ODS-120T (2.15 × 30 cm) was subjected to reverse phase preparative HPLC by gradient elution with an acetonitrile-water system containing 0.1% TFA to obtain the desired F007.
The fractions containing was collected and lyophilized. Obtained F
007 was confirmed to be the target peptide together with the purity by reverse phase HPLC and amino acid analysis.

Example 5 Synthesis of F001 The cysteine residues protected by Acm are at position 7 and B of the A chain.
F001 was prepared in the same manner as in Example 4 except that the chain was at position 7.
Was synthesized. Example 6 Affinity of Insulin Fragment Receptor (2) According to the method described in Example 3, the affinity of the insulin fragments (F007, F001) obtained in Examples 4 and 5 for insulin receptor on capillary endothelial cells. Was measured and it was confirmed that the affinity was similar to the fragments obtained in Examples 1 and 2, respectively. Example 7: Hypoglycemic action of insulin fragment The hypoglycemic action of F007 was compared to the biological activity with insulin by dissolving ICR mice in physiological saline and administering subcutaneously. As shown in Table 3, when the blood glucose level one hour after administration was compared with that of insulin, no remarkable decrease was observed at the same dose, and even if the dose was 10 times the dose, the extreme hypoglycemic effect of insulin was not exhibited. It was This is proof of its usefulness as a carrier peptide, since insulin fragment F007 has a weaker hypoglycemic action and has only brain transport activity as compared with insulin.

[Table 3] Hypoglycemic effect of F007 ICR mouse administration sample Blood glucose level (mg / dl) (n = 5) Mean ± SE ─────────────────────── ──────────── Saline 291 222 175 223 241 230 ± 19 Insulin (30 μg) 113 116 101 108 121 112 ± 3 F007 (30 μg) 190 263 211 218 199 216 ± 13 F007 (300 μg) ) 166 203 158 161 161 170 ± 8 ───────────────────────────────────

Example 8: Interleukin 2 (IL-
2) -Carrier peptide conjugate 7 mg of F007 in 0.1 M phosphate buffer (pH 7.4)
After dissolving in 1 ml and separately prepared SPDP / dimethyl sulfoxide (DMSO) solution (7 mg / 40 μl) was added dropwise little by little under ice cooling, the mixture was allowed to stand at room temperature for 30 minutes. The gel column PD-10 (Pharmacia) was used to remove unreacted SPDP, and this was eluted with 0.1 M phosphate buffer (pH 7.4) and fractionated. On the other hand, 10 mg of recombinant human IL-2 (Takeda Yakuhin) was dissolved in 1 ml of 0.1M phosphate buffer (pH 7.4) in the same manner, and SPDP / DMSO solution (7 mg / 40 mg) prepared separately was dissolved.
μl) was added dropwise little by little under ice cooling, and the mixture was left at room temperature for 30 minutes. PD-10 was used to remove unreacted SPDP, and 0.1 M phosphate buffer (pH 7.4) was used.
Was eluted and collected. This was replaced with a 0.1 M sodium acetate solution (pH 4.5) on PD-10. this,
After concentrating to 2.0 ml under reduced pressure, 3.6% dithiothreitol 0.1 M sodium acetate solution (pH 4.5) was added and the mixture was left standing at room temperature for 30 minutes for reduction. This was collected by using PD-10 to remove small molecules, eluting with 0.1 M phosphate buffer (pH 7.4). Next, SPDP
F007 and IL-2 bound to the above were mixed and allowed to stand for 18 hours, and the resulting reaction solution was separated and purified on a Sephadex G-75 (Pharmacia) column. The obtained F0
The structural formula of 07-IL-2 is shown below.

[Chemical 3]

Example 9: ¹²⁵ I-labeled nerve growth factor (NG)
F) -Carrier peptide conjugate Recombinant human NGF obtained by the method described in JP-A-4-128300 was first labeled with ¹²⁵ I by the chloramine T method. F007 was bound to this using SPDP in the same manner as described in Example 8, and the bound product was separated and purified by reverse phase HPLC, dialyzed against water and lyophilized to give the bound product ( ¹²⁵ I-NGF- F007) was obtained. Example 10: Somatostatin-Carrier Peptide Conjugate A somatostatin-F007 conjugate was obtained in the same manner as in Example 8 using somatostatin in place of IL-2. Example 11: Vasopressin-Carrier Peptide Conjugate A vasopressin-F007 conjugate was obtained in the same manner as in Example 8 using vasopressin instead of IL-2. Example 12: Interferon α-Carrier Peptide Conjugate Example 8 using interferon α in place of IL-2
Interferon α-F007 conjugate was obtained by the same method as described above. Example 13: bFGF-Carrier Peptide Conjugate A bFGF-F007 conjugate was obtained in the same manner as in Example 8 except that bFGF was used instead of IL-2. Example 14: HRP-Carrier Peptide Conjugate A HRP-F007 conjugate was obtained in the same manner as in Example 8 using HRP in place of IL-2.

Formulation Example When the peptide conjugate of the present invention is used as a therapeutic agent for brain disease, it can be administered as the following formulation. 1. 200 mg of the somatostatin-F007 conjugate obtained in Example 10 is dissolved in 10 ml of physiological saline and put in a spray container to give a formulation which can be administered intranasally. 2. 10 mg of the vasopressin-F007 conjugate obtained in Example 11 is dissolved in 10 ml of physiological saline, and 100 mg of methylcellulose is added to prepare a viscous liquid, which is a preparation that can be administered intranasally. 3. Interferon α-F00 obtained in Example 12
10 mg of 7-conjugate is dissolved in 10 ml of physiological saline, and an injection solution for intravenous, subcutaneous and intramuscular administration is prepared according to a conventional method. 4. BFGF-F007 conjugate 1 obtained in Example 13
0 mg is dissolved in 10 ml of physiological saline and an injection solution for intravenous, subcutaneous and intramuscular administration is prepared according to a conventional method.

[0031]

[Sequence list]

 Sequence number (SEQ ID NO): 1 Sequence length (SEQUENCE LENGTH): 21 Sequence type (SEQUENCE TYPE): Amino acid (amino acid) Topology- (TOPOLOGY): Linear type (linear) Sequence type (MOLECULE TYPE) ): Peptide (HIPOTHTICAL): No Sequence features (FEATURE) Location (location): 6 Other features (OTHER INFORMATION): Disulfide bond Location (location): 11 Other features (OTHER) INFORMATION): Disulfide bond sequence (SEQUENCE DESCRIPTION): Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20

Sequence number (SEQ ID NO): 2 Sequence length (SEQUENCE LENGTH): 22 Sequence type (SEQUENCE TYPE): Amino acid (amino acid) Topology- (TOPOLOGY): Linear sequence type (MOLECULE TYPE): Peptide (HIPOTHTICAL): No Sequence characteristics (FEATURE) Other characteristics (OTHER INFORMATION): Sequence (SEQUENCE DESCRIPTION): Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg 20

[Brief description of drawings]

FIG. 1 Insulin fragment F obtained in Example 1
It is a reverse phase HPLC chromatogram of 001.

FIG. 2 Insulin fragment F obtained in Example 1
It is a reverse-phase HPLC chromatogram of 001 and insulin (all molecules).

FIG. 3 Insulin fragment F obtained in Example 1
001 is the estimated structural formula.

FIG. 4 Insulin fragment F obtained in Example 2
It is a reverse phase HPLC chromatogram of 007.

FIG. 5: Insulin fragment F obtained in Example 2
It is a reverse-phase HPLC chromatogram of 007 and insulin (all molecules).

FIG. 6 Insulin fragment F obtained in Example 2
007 is an estimated structural formula.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Reference number within the agency FI Technical display location A61K 37/36 8314-4C C07K 7/10 8318-4H 7/16 8318-4H 7/26 8318-4H 7 / 40 8318-4H 13/00 8318-4H 15/26 8318-4H // C07K 99:00

Claims (16)

[Claims] 1. A blood-brain barrier that is obtained by combining a physiologically active peptide or protein that does not itself cross the blood-brain barrier with a carrier peptide that does not substantially exhibit physiological activity itself and can cross the blood-brain barrier. Peptide conjugates. 2. The carrier peptide has a molecular weight of about 4,000.
2. The conjugate according to claim 1, which is from 100 to 100,000 peptides. 3. The conjugate according to claim 1, wherein the carrier peptide is an inactive fragment of an in vivo peptide capable of passing through the blood-brain barrier by receptor-mediated transcytosis. 4. The conjugate according to claim 2, wherein the in vivo peptide is insulin, transferrin, insulin-like growth factor I or II. 5. The carrier peptide comprises a peptide chain having 14 to 21 amino acid residues from the N terminus of the A chain of insulin and amino acid residues 16 to 22 from the N terminus of the B chain.
The conjugate according to claim 1, which is an insulin fragment consisting of one peptide chain. 6. An insulin fragment is represented by: The conjugate according to claim 5, which is a fragment represented by: 7. The insulin fragment is (B23-3
0) The conjugate according to claim 5, which is an insulin fragment lacking octapeptide. 8. The conjugate according to claim 1, wherein the physiologically active peptide or protein has a molecular weight of about 200 to 120,000. 9. The conjugate according to claim 1, wherein the physiologically active peptide or protein has a molecular weight of about 400 to 80,000. 10. The conjugate according to claim 1, wherein the physiologically active peptide or protein has a molecular weight of about 500 to 60,000. 11. The conjugate according to claim 1, wherein the physiologically active peptide or protein is a neurotrophic factor. 12. The conjugate according to claim 1, wherein the physiologically active peptide or protein is a peptide or protein belonging to the neurotrophin family. 13. The conjugate according to claim 1, wherein the physiologically active peptide or protein is nerve growth factor (NGF). 14. The conjugate according to claim 1, wherein the physiologically active peptide is a neuropeptide. 15. The conjugate according to claim 1, which is obtained by binding a peptide or protein belonging to the neurotrophin family to an insulin fragment. 16. Nerve growth factor (NGF) and (B23-3).
0) The conjugate according to claim 1, which is bound to 0) an octapeptide-deleted insulin fragment.