称号及び氏名

博士（応用生命科学）

学位授与の日付

平成２４年３月３１日

論Development of Novel Drug Delivery System文

名福原 彩乃for Poorly Water-soluble Compounds
Using Lipocalin-type Prostaglandin D Synthase（リポカリン型プロスタグランジン D 合成酵素を用いた難水溶性薬剤に対する新規ドラッグデリバリーシステムの開発）
論文審査委員

主査

乾隆

副査

太田 大策

副査

杉本 憲治

副査

竹内 正 吉論文要旨
Introduction
Most newly synthesized compounds during the drug discovery process are generallyhydrophobic and thus insoluble in water.

Although subsequent chemical modification

improves the solubility of these compounds, their potencies seem to decrease in many cases.
These hydrophobic compounds have frequently been dropped from development in thepreclinical stage. To take advantage of these compounds, special formulations are required toproduce an aqueous dispersion.
Lipocalin-type prostaglandin D synthase (L-PGDS) is an enzyme that catalyses theisomerization of prostaglandin (PG)H2 to PGD2, the major prostanoid produced in the central
nervous system of mammals.

L-PGDS is also a member of the lipocalin gene family, which iscomposed of a group of secretory lipid-transporter proteins, such as retinol-binding protein,β-lactoglobulin, and major urinary protein. A series of biochemical studies of L-PGDS has
revealed that this protein can bind a large variety of hydrophobic small molecules (Mw =200-780) with a dissociation constant (Kd) of 30 nM to 2 µM. The feature defined as ‘broad
ligand selectivity’ suggests some mechanisms by which L-PGDS could be used in a drugdelivery system (DDS) for poorly water-soluble compounds.
Here it is shown the feasibility of a novel DDS using L-PGDS as a delivery vehicle fortwo kinds of poorly water-soluble compounds: diazepam (DZP, Mw = 284.7) and6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, Mw = 336.3).

DZP is a majoranxiolytic

NBQXdrugpossessing

themoietyofbenzodiazepine,

and

is

an

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist.

Inaddition, it is exhibited the relationship between the binding affinities of DZP for L-PGDS andthe efficacy of the complex in vivo.
Chapter 1. The feasibility of DDS for poorly water-soluble drugs using L-PGDS.
In this study the recombinant mouse C65A-substituted L-PGDS mutant, in which a
catalytic residue of cysteine was substituted to alanine to get rid of an enzymatic activity ofL-PGDS, was used.

In order to clarify an affinity and a stoichiometry for the binding of poorly

water-soluble compounds to L-PGDS, the calorimetric analysis on DZP/L-PGDS andNBQX/L-PGDS systems was carried out using isothermal titration calolimetry.

It wasrevealed for DZP and NBQX that each L-PGDS held three molecules with Kd = 47.6 and 16.5µM, respectively.

To confirm the relationship of the compound to L-PGDS, theNBQX/L-PGDS complex was analyzed by mass spectrometry (MS) under non-denaturingconditions.

By MS, the 1:3 complex of L-PGDS and NBQX was observed. Next, I measured

the effect of L-PGDS on the solubility of DZP and NBQX in PBS. L-PGDS of 500 µM
increased the solubility of DZP and NBQX 7- and 2-fold, respectively, compared to PBS alone.
From this, it is clear that L-PGDS can enhance the solubility of poorly water-solublecompounds without using organic solvent.
As it was found that L-PGDS could bind the poorly water-soluble compounds andincrease the solubility of such compounds in vitro, I assessed the effect of L-PGDS to the drug
potencies of DZP and NBQX in vivo. Basically, two delivery strategies were used in theassessment: oral administration of DZP/L-PGDS complex to mice, and intravenous delivery ofNBQX/L-PGDS complex to ischemic gerbils.

First, I investigated the effect of oral

administration of DZP/L-PGDS complex on pentobarbital-induced anesthesia. DZP is known
to intensify pentobarbital-induced anesthesia. DZP was suspended in either distilled watercontaining 0.5% carboxymethylcellulose (CMC), which is widely used as a suspending agent,or L-PGDS solution. As a result, the duration of loss of righting reflex (LORR) in mice treatedwith DZP/CMC suspension (500 µM DZP) was 50% longer than with CMC, and it wascomparable to that of 250 µM DZP/L-PGDS solution.

Mice treated with 500 µM

DZP/L-PGDS solution showed 1.3-fold longer duration of LORR than those treated with 500µM DZP/CMC suspension.

The metabolism of DZP in plasma and forebrain was then

investigated by LC-MS after oral administration of samples.

The result was that theconcentrations of DZP active metabolites such as nordiazepam and oxazepam in DZP/L-PGDS
solution administered groups were higher than in DZP/CMC suspension administered group.
All these results clearly indicate that both the absorption in the gastrointestinal tract and the oral
bioavailability of DZP in the presence of L-PGDS are higher than those in the CMC suspension.
Next, it was investigated the applicability of this DDS to intravenous administration ofNBQX/L-PGDS complexes. The bilateral common carotid arteries were ligated by clip for 5min, followed by removal of the clips and reperfusion. PBS in the presence or absence ofL-PGDS were saturated with NBQX was administered intravenously (100 µl/gerbil) 3 times at 0,10, and 25 min after reperfusion.

One week after reperfusion, the number of surviving

hippocampal CA1 pyramidal cells was counted. The number of pyramidal cells in the control
group decreased to 4% in the sham-operated group. The intravenous injection of saturatedNBQX in PBS did not produce any discernible effects. In contrast, gerbils administered thesame treatment using NBQX/L-PGDS solutions (0.8 mM and 2.0 mM L-PGDS) showedsignificantly increased fractions of living pyramidal cells (17% and 81%, respectively)
compared to the control group.
These results show the feasibility of a novel DDS for poorly water-soluble compounds
such as DZP and NBQX using L-PGDS. This feasibility is also supported by the fact that anL-PGDS-mediated drug delivery was more effective than the delivery of these compoundswithout L-PGDS in both oral and intravenous administration.
Chapter 2. The relationship between the binding affinities of DZP for L-PGDS mutants
and the efficacies of these complexes in vivo.
In chapter 1, I found that L-PGDS can increase the solubility of DZP and the oral
administration of DZP/L-PGDS solution in mice revealed the increased duration ofpentobarbital-induced LORR.

In this chapter, it was investigated the relationship between thebinding affinities of DZP for L-PGDS and the efficacy of intravenous administration of DZPsolubilized by L-PGDS in mice.
First, 21 kinds of L-PGDS mutants were made by site-directed mutagenesis at residues
which are thought to be involved in binding of DZP by NMR titration experiments of L-PGDSwith DZP.

Next, the Kd values of L-PGDS mutants and DZP measured by fluorescencequenching of the intrinsic tryptophan residues of L-PGDS, and the solubility of DZP in eachmutant solution. As a result, the Kd value of L-PGD and DZP was 83.8 µM, while L-PGDS
mutant with the highest binding affinity of DZP (Kd = 2.1 µM) was S81F mutant, in which
Ser81 residue was substituted to phenylalanine, and with the lowest binding affinity (Kd = 341µM) was F83A mutant, in which Phe83 residue was substituted to alanine. In addition, thesolubility of DZP in L-PGDS solution was 361 µM and that in S81F and F83A mutant solution
were 365 and 219 µM, respectively, indicating that L-PGDS mutants possessing high binding
affinity of DZP showed higher solubilities of DZP in their solutions. Further, I investigated theeffect of intravenous administration of DZP/L-PGDS mutant complexes on the duration of thepentobarbital-induced LORR in mice. As a result, the administration of DZP/S81F mutant did
not show significantly longer duration of LORR compared with that of control. On the otherhand, the administration of DZP/L-PGDS and DZP/F83A mutant solution showed 1.4- and1.7-fold longer duration of LORR, suggesting that the lower affinity of L-PGDS mutant for DZP,
thehigher

thepotency

intravenously-administered.

ofDZP

when

DZP/L-PGDSmutantsolutions

wereThese results indicated that a balance of the binding affinity ofL-PGDS and DZP and the solubility of DZP in L-PGDS solution is becoming a really key point.
Conclusions
In this study, it was shown that L-PGDS could improve the solubility of DZP andNBQX in aqueous solutions, and consequently collected evidence which revealed theprospective effect of each compound in vivo. In addition, it was identified the relationshipbetween the binding affinity of L-PGDS and DZP and the drug potency of DZP solubilized byL-PGDS.

Further investigation using L-PGDS mutants with different binding affinities forother drugs would be able to engineer a tailor-made carrier protein possessing
molecular-selective recognition for several hydrophobic drugs using L-PGDS as a template.
These results demonstrate that L-PGDS is a beneficial delivery vehicle for poorly water-solublecompounds. This novel DDS will facilitate the pharmaceutical and clinical developments ofvarious water-insoluble compounds.

審査結果の要旨
近年，医薬品に対する安全性が厳しく求められているために，創薬現場では高度なバイオサイエンス技術が要求され，新薬開発コストの急騰を招いている。一方，薬剤が難水溶性であることや，薬剤溶解度向上を図るための化学修飾による薬剤活性の低下により，開発中止になった薬剤候補を，復活させる動きが世界的に始まっている。これらのニーズを実現させるためには大きなブレークスルーが必要であり，その本命としてドラッグデリバリーシステム（DDS）が著しい発展を遂げている。本研究の目的は，生体内輸送蛋白質を用いて難水溶性薬剤に対する新規 DDS を開発し，その概念検証を in vivoレベルで行うことである。DDS キャリアとして，リポカリン型プロスタグランジン D合成酵素（L-PGDS）を用い，難水溶性薬剤として，ベンゾジアゼピン系抗不安薬である diazepam（DZP）と AMPA 型グルタミン酸拮抗薬である 6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione（NBQX）を用いた。
第１章では，L-PGDS と薬剤の相互作用，構造解析，及び in vivo における薬剤複合体の効果を調べている。等温滴定型熱量測定により，L-PGDS は DZP，あるいは NBQXを 3 分子結合し，その解離定数（Kd）は，それぞれ 47.6 µM，及び 16.5 µM であることを示した。また，非変性条件下における NBQX/L-PGDS 複合体の質量分析により，
L-PGDS は確かに 3 分子の NBQX を結合できることが明らかとなり，熱量測定の結果と一致した。また，X 線小角散乱，及び NMR 測定により，L-PGDS は薬剤をその疎水性ポケット内部に結合し，コンパクトになることが分った。さらに，L-PGDS 存在下における DZP，及び NBQX の濃度は，L-PGDS の濃度依存的に上昇することが判明した。
次に，本 DDS の in vivo による評価を行うために，ペントバルビタール麻酔下のマウスに DZP/L-PGDS 複合体を経口投与し，DZP の効果を麻酔時間の延長を指標として調べた。その結果，DZP/L-PGDS 複合体を投与したマウスは，対照実験としてカルボキシメチルセルロース（CMC）に懸濁した DZP を投与したマウスと比較して，麻酔時間の有意な延長が観測された。また，液体クロマトグラフィー法により，DZP/L-PGDS 複合体の経口投与後の血中，及び脳内の DZP 活性代謝物の濃度を測定した結果，DZP/CMC 懸濁液を投与したマウスに比べて上昇していた。これらの結果は，CMC と比較して，L-PGDS が DZP のバイオアベイラビリティを上昇させたことを示唆している。さらに，
スナネズミ脳虚血モデルにおいて，NBQX/L-PGDS 複合体の静脈内投与による，海馬CA1 領域の遅発性神経細胞死への影響を調べた。NBQX のみの投与群は，NBQX が難水溶性であるため PBS にほとんど溶解せず，対照群と変わらなかった。しかし，
NBQX/L-PGDS 複合体の投与群では，遅発性神経細胞死が有意に抑制された。以上の結果は，難水溶性薬剤に対する本 DDS が，経口投与，及び静脈内投与の両投与法において有用であることを示している。
第２章では，難水溶性薬剤/L-PGDS 複合体の静脈内投与における，薬剤と L-PGDSの結合親和性の影響を調べている。まず，部位特異的変異により作製した 21 種類のL-PGDS 変異体と DZP の結合親和性をトリプトファン蛍光消光実験により調べた。また，変異体溶液存在下における DZP 濃度を測定した。その結果，DZP に対する結合親和性が高い変異体ほど，溶液中の濃度が上昇することが判明した。さらに，DZP/変異体複合体のマウス静脈内投与による，ペントバルビタール誘導性麻酔時間への影響を調べた
結果，DZP に対する結合親和性が低い複合体ほど，麻酔時間が延長し，DZP の効果が上昇することが判明した。以上の結果は，薬剤の性状に応じたキャリアを作製すること，つまり様々な難水溶性薬剤に応じたテーラメード DDS キャリアの作製が可能であることを示すものである。
本研究は，生体内輸送蛋白質である L-PGDS を用いることにより，十分な薬効を持つにもかかわらず，難水溶性のために開発段階で除外されてきた薬剤の臨床使用への可能性を初めて示したものであり，これら薬剤の臨床応用を目指す上で非常に重要な知見であると言える。また，蛋白質を用いた難水溶性薬剤に対する DDS は世界的にも稀有であり，学術的新規性，及び独創性が高い。よって，最終試験の結果とあわせて，博士（応用生命科学）の学位を授与することを適当と認める。