JP2013224283A - Peptide indicating renal clustering, drug carrier, drug formulation, and pharmaceutical composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide indicating clustering specifically to the kidney, a drug carrier and a pharmaceutical composition containing such peptide.SOLUTION: A peptide indicating renal clustering is characterized by including a specific amino acid sequence. It is confirmed experimentally that the peptide does not indicate specific clustering with respect to the heart, the liver, and the lungs but indicates specific clustering only with respect to the kidney. By introducing such peptides into a carrier or a drug, the carrier or the drug may be clustered specifically only to the kidney.

Description

  The present invention relates to a peptide that specifically accumulates in the kidney. The invention also relates to drug carriers and pharmaceutical compositions containing such peptides.

  Many drugs are delivered to the target organ or tissue through the blood circulation. In most cases, however, the drug is not only targeted to the diseased organ or tissue, but is also taken up by other organs and tissues in the body. As a result, undesirable side effects may occur in the patient. For this reason, it is desirable to selectively target specific organs or tissues to deliver drugs.

  A drug delivery system (DDS) is a drug delivery system designed to show pre-set drug release and tissue reach in vivo for drug delivery optimization and has already been commercialized. There is something that has been done. As a drug carrier for DDS, a protein or peptide has been sought using a phage display method, but low selectivity for drug transfer to the kidney is regarded as a problem (Patent Document 1, Non-Patent Documents 1 to 3). 2).

  Coupling a drug carrier (eg, a viral vector or albumin) with a targeting molecule can improve drug uptake into the target organ or tissue without affecting the chemical and physical properties of the drug. It is considered possible. When peptides are used as targeting molecules, the conformation of the carrier is less likely to change due to the low molecular weight, and it is easier to couple to a carrier or drug than a protein with a large molecular weight.

  Patent Document 2 discloses a peptide that specifically binds to endothelial cells. The peptides are said to be incorporated into gene delivery vehicles and can lead to therapeutic agents including proteins (such as growth factors and cytokines) and small molecules (such as drugs and other therapeutic agents). The target vector, peptide, or small molecule disclosed in Patent Document 1 is used for cancer, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, platelet destruction, restenosis, ischemic vascular disorder, wound healing, congestive heart failure Can be used to treat various diseases such as heart disease such as ischemic heart disease, peripheral limb disease, vein transplant stenosis, and restenosis, myocardial ischemia, reperfusion injury, peripheral arterial disease, obesity It is supposed to be possible.

  Patent Document 3 discloses a composition comprising a targeting peptide that selectively binds to endometriosis cells. The targeting peptide disclosed in Patent Document 2 can be administered in vivo in a pharmaceutically acceptable carrier. It is also disclosed that the provided compositions can further comprise a pharmaceutically acceptable carrier.

  On the other hand, with the increase in chronic kidney disease against the background of an aging society, development of a new drug therapy is eagerly desired, but the selectivity of drugs for the kidney is regarded as a problem. For this reason, a targeting element for actively transporting drugs to the kidney is essential.

  Non-Patent Document 1 discloses a renal accumulation type peptide having an amino acid sequence of Cys-Leu-Pro-Val-Ala-Ser-Cys. Non-Patent Document 2 discloses a renal accumulation peptide having an amino acid sequence of His-Thr-Thr-His-Arg-Glu-Pro. Non-Patent Documents 3 and 4 disclose a method for preparing a phage display library and an in vitro screening method.

International Publication No. 2008 / 084899A1 Pamphlet JP-T-2006-510360 Special table 2009-521512

Renata. Pasquailini, E. Ruoslahti, Nature, Vol.380, 364-366, (1996) Laura Denby, Lorraine M. Work, Delyth Graham, Catherine Hsu, Dan J. Von Seggern, Stuart A. Nicklin, and Dr. Andrew H. Baker, Human Gene Therapy. November 2004, Vol. 15, No. 11: 1054- 1064 Scott, J.M. and Smith, G.P., Science, 249, 386-390 (1990) Smith, G.P. and Scott, J.K., Methods in Enzymology, 217, 228-257 (1993)

  Since the peptide disclosed in Non-Patent Document 1 has not been evaluated for its ability to accumulate in other organs, it is unclear whether it specifically accumulates in the kidney. In addition, the peptide disclosed in Non-Patent Document 2 shows about 5-fold accumulation by introducing three peptides into a carrier, but it is about 1.7-fold per peptide, and the accumulation is high. I can't say that. For this reason, it is necessary to search for peptides that specifically accumulate in the kidney.

  An object of the present invention is to provide a peptide that specifically accumulates in the kidney, and a drug carrier and a pharmaceutical composition containing such a peptide.

  As a result of searching for peptides that specifically accumulate in the kidney from the phage library by the in vivo phage display method, the present inventors found three types of peptides that specifically accumulate in the kidney, and found the present invention. It came to complete.

  Specifically, the present invention relates to a peptide having an amino acid sequence represented by any of SEQ ID NO: 2, 3 or 5.

  Each of the peptides of the present invention is a cyclic peptide composed of 9 amino acids whose both ends are Cys.

  The present invention also relates to a renal accumulation drug carrier comprising a peptide having the amino acid sequence shown in any of SEQ ID NOs: 2, 3 or 5 and a carrier.

  The present invention also relates to a preparation containing the peptide and the drug.

  The present invention also relates to a pharmaceutical composition comprising the drug carrier and a drug to be delivered to the kidney.

  When the peptide of the present invention is introduced into a carrier or drug, the carrier or drug can be specifically accumulated only in the kidney.

The recovery amount per 1 g of kidney of the phage containing the peptides shown in SEQ ID NOs: 1 to 5 is shown. The recovered amount per 1 g of the heart of the phage containing the peptides shown in SEQ ID NO: 2, 3 and 5 is shown. The recovery amount per 1 g of liver of the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5 is shown. The recovered amount per 1 g of lung of the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5 is shown. It is the fluorescence micrograph of a renal tubule, (a) is after the peptide administration of sequence number: 2, (b) shows the fluorescence micrograph after control peptide administration.

  Embodiments of the present invention will be described with reference to the drawings as appropriate. The present invention is not limited to the following description.

(Drug carrier)
Specific examples of the drug carrier used in the present invention include viral vectors (retroviral vectors, AAV vectors and modified or chemically modified products thereof), albumin, natural polymers, synthetic polymers, liposomes, emulsions, micelles, microcells. The sphere or nanosphere is not particularly limited as long as it does not affect the chemical and physical properties of the drug and can be applied as a drug transportation means.

  Specific examples of drugs to be delivered to the kidney include pharmaceutical compounds such as anticancer drugs, interferons, antibodies, antibacterial drugs, antiviral drugs, antifungal drugs, antiparasitic drugs, diuretics, steroid drugs, immunosuppressive drugs, anti Although it is a platelet drug or an antithrombotic drug, it is not particularly limited as long as it is a drug having a therapeutic effect on kidney disease. A pharmaceutical composition can be produced by, for example, chemically linking a peptide of the present invention (any peptide shown in SEQ ID NO: 2, 3 or 5) and a drug. When the drug is a protein, a pharmaceutical composition can be produced by using the drug and the peptide of the present invention as a fusion protein.

  Specific examples of the anticancer drug include sorafenib, sunitinib, cyclophosphamide, melphalan, ranimustine, ifosfamide, nitrogen mustard-N-oxide, 6-mercaptopurine, riboside, enocitabine, carmofur, cytarabine, cytarabine ocphosfephene Wheat, tegafur, 5-FU, doxyfluuridine, doxyfluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, epirubicin hydrochloride, doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin hydrochloride, pleomycin hydrochloride, Dinostatin styramer, bleomycin sulfate, mitomycin C, neocartinostatin, pepromycin sulfate, etoposide, irino hydrochloride Tecan, docetaxel hydrate, vincristine sulfate, vindesine sulfate, vinblastine sulfate, paclitaxel, acegraton, ubenimex, cisplatin, schizophyllan, sobuzoxan, krestin, toremifene citrate, medroxyprogesterone acetate, tamoxifen citrate, carboplatin hydrochloride hydrate , Procarbazine hydrochloride, mitoxantrone hydrochloride, L-asparaginase, tretinoin, nedabratine, picibanil, flutamide, pentostatin, porfimer sodium, or lecithinan.

  For the identification of peptides that specifically accumulate in the kidney, it is preferable to employ a phage display method. Phage display methods are used to express a large number of peptides that can be screened in vitro using a particular target molecule or cell to identify peptides that specifically bind to the target molecule or cell. . Regarding the method for preparing the phage display library and the in vitro screening method, the methods disclosed in Non-Patent Documents 3 and 4 can be used.

<Construction of phage library displaying random peptide sequence of Cys-Xaa-Cys>
A phage displaying a random peptide sequence of Cys-Xaa-Cys on the surface was prepared by inserting the random sequence into a fUSE5 vector. Xaa means 19 kinds of normal amino acids.

<Screening of kidney accumulation phage by in vivo phage display method>
Wistar male rats (6 weeks old, body weight 170 g to 200 g) were intravenously injected with 10 ¹⁰ TU of Cys-Xaa-Cys phage under pentobarbital anesthesia. After 15 minutes, the whole body was bled by refluxing PBS from the heart, and then the kidney was removed. The removed kidney was weighed after the renal capsule was peeled off in Lysis buffer. Thereafter, the kidney from which the kidney membrane was peeled was homogenized, and the obtained kidney homogenate solution was centrifuged (2000 rpm, 4 ° C., 10 minutes). Furthermore, the supernatant was applied to an ultracentrifuge (55000 rpm, 4 ° C., 13 minutes). The obtained pellet was used as a kidney-accumulating phage.

The pellet obtained by ultracentrifugation was suspended using 500 μL of PBS. 2 mL of E. coli (OD ₆₀₀ = 0.18 to 0.2) was added and incubated at room temperature for 1 hour to infect the phage in the pellet with E. coli. Then, add LB medium (containing kanamycin and tetracycline), make the total volume 10mL, measure the amount of phage recovered from this part (TU), incubate the rest (230rpm, 37 ° C, 16 hours), Phages were grown.

  After completion of the incubation, the mixture was centrifuged (8000 rpm, 4 ° C., 20 minutes), and 1.5 mL of PEG was added to the supernatant. Phage was salted out by cooling on ice for 1 hour, and a phage pellet was obtained by centrifugation (8000 rpm, 4 ° C., 20 minutes). In order to improve the purity of the phage, the pellet was suspended again in 1.5 ml of PBS and then centrifuged (10000 rpm, 4 ° C., 5 min). After adding 225 μL of PEG to the supernatant and cooling on ice for 10 minutes, a phage pellet was obtained by centrifugation (10000 rpm, 4 ° C., 5 minutes). The obtained pellet was suspended using 500 μL of PBS to obtain a candidate phage solution. The operation from the phage administration to the phage solution was repeated 3 times to screen for kidney-accumulating phage.

  E. coli was infected with the kidney-accumulating phage screened by the above operation to form colonies. The formed colonies were each dissolved in a 10% glycerin aqueous solution to prepare a colony solution. Thereafter, PCR (1: 96 ° C., 5 minutes (1 cycle), 2: 96 ° C., 10 seconds / 50 ° C. using BigDye® Terminator v3.1 Cycle Sequencing Kit and fUse5 Reverse Primer (1.6 pmol / μL) The phage DNA was grown for 5 seconds / 60 ° C., 4 minutes (25 cycles), 3: 4 ° C., overnight).

  1 μL of 3M sodium acetate and 25 μL of 99.5% ethanol were added to the phage DNA, which is a PCR product, and cooled on ice for 10 minutes. Thereafter, the mixture was centrifuged (15000 rpm, 20 ° C., 20 minutes), and 70 μL of 70% ethanol was added to the obtained pellet. The solution was further centrifuged (15000 rpm, 20 ° C., 5 minutes), and the resulting pellet was vacuum dried. After the drying, 12 μL of Hi-Di formamide reagent (Applied Biosystems) was added. After incubating at 95 ° C. for 2 minutes, the mixture was further cooled on ice for 2 minutes. 4 μL of the sample solution thus obtained and 8 μL of Hi-Di formamide reagent were placed in a tube for capillary electrophoresis and mixed. The mixed solution was subjected to capillary electrophoresis to analyze the amino acid sequence of the peptide possessed by the kidney-accumulating phage.

<Peptide sequence analysis of recovered phage>
The peptide sequence of the collected phage was analyzed using ABI310 genetic analyzer.

  Table 1 shows the peptide sequence analysis of the recovered phage. In Table 1, “Number” means the number of obtained clones, and “%” means a ratio (%) to the total number of clones.

  From Table 1, since a plurality of clones were confirmed for each of the peptides of SEQ ID NOs: 1 to 5, in vivo experiments using mice were further conducted on the renal accumulation properties of these five types of peptides.

<Evaluation of kidney accumulation of 5 types of phage>
(1) Evaluation test based on the number of colonies
Five types of peptides (C-ILASGVT-C, C-HGVQARL-C, and C-ILASGVT-C in amino acid one-letter code) were screened by phage display method under anesthesia with pentobarbital in male Wistar rats (6 weeks old, 170g-200g) -RIGGMSN-C, C-SSYSSVT-C, and C-VMATGGV-C / that is, the peptides displayed in SEQ ID NOs: 1 to 5) were dissolved in PBS, and 10 ¹⁰ TU was injected into the tail vein. did. After 15 minutes, whole body blood was removed by refluxing PBS-tween 0.05% at 42 ° C. using a peristaltic pump, and each organ was removed. The excised organ was washed in Lysis buffer and then weighed. Thereafter, phages accumulated in each organ were collected by obtaining a homogenate solution of each organ by homogenization.

Thereafter, 2 mL of E. coli (OD ₆₀₀ = 0.18 to 0.2) was added to the homogenate solution of each organ and incubated at room temperature for 1 hour. Thereafter, 5 mL of LB medium (containing kanamycin and tetracycline) was added. The obtained solution was spread on 100 μL of LB medium (containing kanamycin and tetracycline), and the amount of phage accumulated per gram of each organ was calculated based on the number of colonies formed and the organ weight, and the accumulation was evaluated. . As a control phage, Fd-tet (peptide insertless phage) was used.

  FIG. 1 shows the amount recovered per 1 g of kidney of a phage containing the peptides shown in SEQ ID NOs: 1 to 5. The numbers on the horizontal axis of the graph indicate the sequence numbers. As shown in FIG. 1, the phages containing the peptides shown in SEQ ID NOs: 2, 3 and 5 show a significantly higher recovery amount and higher accumulation with respect to the kidney than the control phage recovery amount. confirmed. In particular, the amount of peptide accumulated in the kidney shown in SEQ ID NO: 2 was about 20 times that in the control phage.

  FIG. 2 shows the recovered amount per 1 g of the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5. The numbers on the horizontal axis of the graph indicate the sequence numbers. Compared with the recovered amount of control phage, the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5 did not show any specific accumulation in the heart.

  FIG. 3 shows the recovery amount per 1 g of liver of the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5. The numbers on the horizontal axis of the graph indicate the sequence numbers. Compared with the recovered amount of control phage, the phages containing the peptides shown in SEQ ID NOs: 2, 3 and 5 did not show any specific accumulation in the liver.

  FIG. 4 shows the recovered amount per 1 g of lung of the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5. The numbers on the horizontal axis of the graph indicate the sequence numbers. Compared with the recovered amount of control phage, the phage containing the peptides shown in SEQ ID NOs: 2, 3 and 5 did not show any specific accumulation in the lung.

(2) Evaluation test by fluorescence microscope observation According to the evaluation test of (1) above, C-HGVQARL-C (the peptide shown in SEQ ID NO: 2), which showed the most accumulation in the kidney, and the accumulation in the kidney C-SSYSSVT-C (peptide shown in SEQ ID NO: 4), which was not found, was synthesized by entrusting a peptide (FITC-labeled peptide, purity 90%) with FITC bound to the N-terminus of both peptides to BEX. did. The peptides of SEQ ID NOs: 2 and 4 were synthesized using a peptide synthesizer (Shimadzu Corporation, PSSM-8).

  Two types of FITC-labeled peptides were dissolved in DMSO and adjusted to a concentration of 20 mM. Thereafter, 200 μL of a peptide solution diluted 20-fold with PBS was intravenously injected into Wistar male rats (6 weeks old, 170 g to 200 g) under pentobarbital anesthesia, respectively. After 5 minutes, systemic blood removal was performed by refluxing PBS at 42 ° C. using a perista pump. Thereafter, each organ was removed. The excised organ was washed in Lysis buffer and then fixed by being immersed in 4% paraformaldehyde solution for 12 hours. The fixed organ was embedded in paraffin by a tissue specimen preparation system (Milestone). Thereafter, a 4 μm section was cut out from each embedded organ.

  The prepared sections were immersed in 100% ethanol to remove paraffin, and then encapsulated with marinol. And each structure | tissue was observed using the fluorescence microscope (excitation light 470nm).

  FIG. 5 is a fluorescence micrograph of renal tubules, (a) after administration of the FITC-labeled peptide of SEQ ID NO: 2, and (b) fluorescence microscope after administration of the FITC-labeled SEQ ID NO: 4 peptide. Each photo is shown. As shown in FIG. 5 (a), fluorescence was observed in the renal tubule after administration of the peptide of SEQ ID NO: 2 labeled with FITC. However, as shown in FIG. 5 (b), no fluorescence was observed in the renal tubules after administration of the peptide of SEQ ID NO: 4 labeled with FITC. From this, it was confirmed that the peptide of SEQ ID NO: 2 was specifically taken up into kidney cells.

  On the other hand, no fluorescence was observed for heart, liver and lung cells when any of the two types of FITC-labeled peptides was administered.

  As described above, it was confirmed that the peptides of SEQ ID NOs: 2, 3 and 5 did not show accumulation in other organs and specifically showed accumulation in only the kidney. By binding these peptides to a drug carrier such as a viral vector or albumin or to a drug molecule such as doxorubicin hydrochloride or vincristine sulfate, the drug can be specifically incorporated into kidney cells. It was inferred that

  The present invention is particularly useful in the pharmaceutical field.

Claims (4)

  A peptide having the amino acid sequence shown in any one of SEQ ID NOs: 2, 3 and 5.   A kidney-accumulating drug carrier comprising the peptide according to claim 1 and a carrier.   A preparation comprising the peptide according to claim 1 and a drug.   A pharmaceutical composition comprising a drug carrier according to claim 2 and a drug to be delivered to the kidney.