JP5529609B2 - Drug carrier having a target binding part - Google Patents

Description

  The present invention relates to a drug transporter that selectively transports a poorly water-soluble drug to a target place, site, or cell in a living body.

  Drug candidate compounds that act on target receptors discovered by genome drug discovery research generally have a large molecular weight and low solubility in water. In addition, many of the currently used drugs are highly hydrophobic and poorly water-soluble. Although water solubility is increased by chemical modification methods, drug activity is often reduced. If a poorly water-soluble drug having high drug activity can be efficiently transported to the diseased part, clinical application examples of the poorly water-soluble drug can be expanded, and the drug solubility problem that has been difficult for pharmaceutical companies can be solved.

  The key to this issue is the drug delivery system that can solubilize poorly water-soluble drugs, transport them to their targets, and efficiently express their functions in cells. (DDS) development.

  Some of these DDS ideas have already been disclosed. Patent Document 1 discloses a complex including a target binding component, a cavity forming component, and a pharmacological compound, wherein the pharmacological compound is present in the cavity of the cavity forming component. The use of the complex disclosed herein is a protein having a cavity that specifically binds to a target such as a receptor expressed by a subject (particularly, NGF family, interleukin, GM-CSF, EGF, FGF, barnase). , T4 lysozyme, TGFb, IgG, etc.), and a drug that can actually be occluded is selected from drugs having a drug effect on the subject in the cavity.

JP 2000-507210 A

  However, in Patent Document 1, although neuroblastoma is the target, the target overexpressed is the trkA receptor, and NGF is shown as a protein having a target binding component and a cavity-forming component. No concept is disclosed, and the concept of what is stored and how NGF is occluded is merely shown.

  Therefore, it cannot be said that a DDS system that solubilizes a poorly water-soluble drug and mails it to a diseased part to exert the drug efficacy is still provided. In view of such circumstances, the present invention has been conceived for the purpose of developing a DDS system in which a poorly water-soluble drug is solubilized and mailed to a diseased part.

  The present invention creates a drug carrier (also called intelligent artificial protein (iAP)) having both a poorly water-soluble drug delivery function and a cancer cell recognition function, and proposes clinical application. And the lipocalin type | mold prostaglandin D synthase (L-PGDS) which is a biological transport protein is used as a template protein of the main-body part of a chemical | medical agent carrier. That is, the present invention provides a protein having a target binding moiety linked to the N-terminus or C-terminus of lipocalin-type prostaglandin D synthase.

  Lipocalin protein is a transport protein that binds and transports hydrophobic physiologically active substances such as retinoids, thyroid hormones, sex hormones and heme metabolites, and has high binding affinity for hydrophobic small molecules, making it difficult for artificial drugs such as drugs. It can be a water-soluble low-molecular transporter.

  As the drug transportability, by introducing a mutation into the amino acid in the hydrophobic pocket of L-PGDS, the drug to be used is tailored, and the difference in binding affinity is utilized for each drug. Retention characteristics can be adjusted.

  On the other hand, as for the binding portion, when targeting cancer cells or the like, a peptide sequence (Asp-Gly-Arg (NGR)) that specifically binds to a membrane protein (CD13) expressed in neovascular endothelial cells. -Motif) is introduced into the N-terminus of L-PGDS and targeted to cancer tissue.

  Lipocalin-type prostaglandin D synthase is an in-vivo product and can easily overcome the problems generally associated with DDS such as rejection and toxicity. In addition, the amino acid sequence has already been analyzed and can be easily produced by carrying out recombinant synthesis including the target binding site for the target. As described above, the drug carrier of the present invention has a molecular selective recognition function, enables creation of a “nanocarrier protein” that targets cancer tissue, and can provide a new direction in the field of drug development.

It is a conceptual diagram which shows the chemical | medical agent carrier of this invention. It is an illustration of the lipocalin family. It is a figure which shows the detailed structure of a lipocalin type prostaglandin D synthase. It is the graph which dissolved SN-38 in L-PGDS and investigated anticancer activity.

  In FIG. 1, the conceptual diagram of the chemical | medical agent carrier (intelligent artificial protein) of this invention is shown. The drug carrier 1 includes a lipocalin-type prostaglandin D synthase portion 2 as a main body and a target binding portion 4. The lipocalin type prostaglandin D synthase portion 2 which is the main body can utilize a lipocalin protein known as the lipocalin family.

  As will be described later, the lipocalin family has a pocket with a barrel structure, which is shaped like a cup. Here, the slightly water-soluble medicine 6 can be stored. The lipocalin-type prostaglandin D synthase portion 2 has a target binding portion 4, and this portion binds to the target to transport the drug 6 to the target site.

  The lipocalin protein belonging to the lipocalin family is a protein having 160 to 200 amino acid residues and a molecular weight of about 20,000. Moreover, it is known that the structural similarity is very high even though the homology of amino acid sequences is about 20%.

  FIG. 2 shows a part of the lipocalin family. Retinol binding protein, major urine protein, villin binding protein, β-lactoglobulin, lobster crustocyanin, odorant binding protein, etc. are members of the lipocalin family.

  These folding structures of lipocalin proteins are composed of eight antiparallel β sheets that are continuously hydrogen bonded, and have a barrel shape surrounding the hydrophobic low molecular (ligand) binding portion. Seven loops connecting eight β sheets have six short hairpins and one omega loop, and this omega loop is considered to serve as a lid for the ligand binding part. It also has one α helix. That is, the structural similarity of these lipocalin families is that they have eight barrel structures, one omega loop, and one α helix.

  FIG. 3 shows this structure in more detail. FIG. 3 is a schematic diagram of the crystal structure of lipocalin-type prostaglandin D synthase (L-PGDS). It has eight barrel structures (11 to 18), one α helix (19) and one omega loop (H2 helix) (20).

In the first place, L-PGDS is an enzyme that synthesizes PGD ₂ using PGH ₂ synthesized from arachidonic acid as a substrate by the action of cyclooxygenase. In the central nervous system, PGD ₂ exhibits actions such as sleep induction, hypothermia, suppression of progesterone secretion, pain and odor response regulation, and in peripheral nerves, it is released as a mediator of allergy from mast cells, vasodilation, It is a substance that exhibits actions such as bronchoconstriction and inhibition of platelet aggregation.

  L-PGDS also belongs to the lipocalin family, and the pocket part formed by the eight barrel structure has a structure in which a hydrophobic group is arranged on the inside. It can be dissolved in water.

  Regarding the water solubility of a poorly water-soluble drug, a mutation may be introduced into the amino acid inside the pocket to adjust the retention property of the drug in the pocket for each drug. Since L-PGDS is an enzyme in the first place, it is necessary to deactivate the enzyme activity when used as DDS. For this purpose, the 43rd cysteine (active center) from the N-terminus is substituted with alanine to deactivate the enzyme activity.

  About a target coupling | bond part, it can design according to a target. For example, in the case of cancer cells, a peptide sequence NGR that specifically binds to a membrane protein (CD13) expressed in neovascular endothelial cells can be selected. Alternatively, an isoAsp-Gly-Arg (isoDGR) motif that recognizes αvβ3 integrin may be introduced into the N-terminus or C-terminus of L-PGDS by genetic recombination.

  In addition, Lys-Leu-Pro (KLP) -motif (Cancer Res, 97, 1075-81, 2006) for recognizing peritoneal tumor of gastric cancer, Asn-Val-Val-Arg-Gln for recognizing metastatic cancer cells ( NVVRQ) motif (Clin Cancer Res, 14, 5494-502, 2008), Phe-Gln-His-Pro-Ser-Phe-Ile (FQHPSFI) motif (Mol Med, 13, 246-54) that recognizes liver cancer cells. , 2007) can also be suitably used.

  This target binding part is preferably introduced at the end of the lipocalin type prostaglandin D synthase part which is the main body. Further, the N-terminus is more preferable because it is easier to produce a recombinant.

  As the drug that can be transported to the drug carrier of the present invention, a commercially available poorly water-soluble anticancer drug can be suitably used. For example, procarbazine (brain tumor), lomustine (brain tumor), irinotecan (colon cancer), chlorambucil (ovarian cancer), paclitaxel (breast cancer), tamoxifen (breast cancer) and the like.

  The drug carrier of the present invention can be obtained as a recombinant together with the target binding part. The method for producing the recombinant may be in accordance with ordinary methods. That is, a cDNA encoding L-PGDS is obtained by cloning, amplified, and an insert is prepared. This insert is ligated with a separately prepared vector.

  The vector thus obtained is transformed into E. coli, and E. coli is transformed. This Escherichia coli can be cultured to obtain the drug carrier of the present invention.

  The effect of L-PGDS on poorly soluble drugs was confirmed. 7-Ethyl-10-hydroxy-camptothecin (7-Ethyl-10-hydroxy-camptothecin: hereinafter referred to as “SN-38”) is a quinoline alkaloid isolated from the bark and trunk of Chinese cynomolgus, which is native to China. It is known as a topoisomerase I inhibitor. Inhibition of topoisomerase I prevents DNA recombination and leads to apoptosis. For this reason, SN-38 has anticancer activity. However, since SN-38 is hardly soluble, irinotecan hydrochloride (CPT-11), which is a similar substance, is clinically used. Irinotecan is a so-called prodrug that becomes SN-38 and becomes active when hydrolyzed.

  However, the metabolic conversion efficiency of irinotecan is 10% or less of the administered amount, and the efficiency is low. If SN-38 can be dissolved directly and administered to the affected area, it is considered to be a useful means for greatly promoting cancer treatment. Therefore, SN-38 was solubilized using L-PGDS, and its antitumor effect was examined.

<Preparation of drug solution>
A SN-38 / L-PGDS solution (490 μM SN-38) was prepared by sufficiently suspending a large excess of SN-38 in PBS containing 2 mM L-PGDS and passing through a 0.2 μm filter to remove insoluble particles. did. L-PGDS abolished the enzyme activity.

<Measurement of antitumor activity>
The human colon cancer cell line DLD-1 was subcutaneously administered to the flank of a 7-week-old female BALB / c nude mouse at 5 × 10 ⁶ cells. The tumor volume was calculated from the formula: tumor volume = (major axis × minor axis ² ) / 2. When tumor volume reached 200 mm ³ , SN-38 / L-PGDS solution (2 animals) or L-PGDS solution (1 animal, 150 μl each) as a control on days 0, 3, 6, 9 Administered directly to the tumor.

The results are shown in FIG. The horizontal axis represents the number of days (day) since the start of administration, and the vertical axis represents the tumor volume (mm ³ ) determined from the above formula. The vertical triangle mark indicates the day when the SN-38 / L-PGDS solution or L-PGDS solution was administered. Moreover, the tumor volume of the mouse | mouth which administered SN-38 / L-PGDS was shown by the black triangle and the black square, and the mouse | mouth which administered only L-PGDS was shown by the black circle.

  Starting from the same tumor volume and already on day 1 after administration, the tumor volume of mice administered SN-38 / L-PGDS was smaller than that of mice administered L-PGDS alone. Thereafter, the tumor volume of the mice administered with N-38 / L-PGDS hardly increased, and the tumor volume of the two mice increased by a similar level until day 9. On the other hand, mice administered with L-PGDS alone had only a slight increase from the second day to the fourth day, but thereafter the tumor volume increased day by day.

  As described above, the growth of tumor volume was suppressed in the mice (black triangles, black squares) administered with the SN-38 / L-PGDS solution compared to the mice (black circles) administered with the L-PGDS solution. From this result, it became clear that L-PGDS solubilizes SN-38, and that SN-38 solubilized by L-PGDS exhibits an antitumor effect in vivo.

  The drug carrier of the present invention can be used not only for transporting drugs to target cells in vivo but also in vitro.

1 Drug Carrier 2 Body 4 Target Binding Part 6 Drug 11-18 Barrel 19 α Heliox 20 Omega Loop (H2 Helix)

Claims (1)

A drug carrier in which SN-38 is dissolved in a synthetic product in which a peptide sequence of Asp-Gly-Arg is added to the N-terminus of a lipocalin-type prostaglandin D synthase .