Drug Delivery Systems

WORLD-WIDE LEADER in

DDSPEG DERIVATIVES, PHOSPHOLIPIDS AND DRUG DELIVERY MATERIALS

FOR PHARMACEUTICAL PRODUCTS AND FORMULATIONSPEG DERIVATIVES, PHOSPHOLIPIDS AND DRUG DELIVERY MATERIALS

FOR PHARMACEUTICAL PRODUCTS AND FORMULATIONS

WORLD-WIDE LEADER in

Drug Delivery Systems

CATALOGUE Ver.8

DDS

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Table of Contents

I. High Quality PEGs and Activated PEGs

II. Phospholipids derivatives and

Liposome Formulation

III. High-purity Polysorbate 80

IV. High-purity Oleic Acid Derivatives

V. High-purity low molecular weight MACROGOL

VI. Cholesterol Pullulan

VII. MPC Polymers

VIII. Hydrophobic Drug solubilization Kit

IX. High purity CHITOSANs for DDS

X. Biocompatible PEG Anchors for cell membrane

XI. LIPONIZER® for Liposome production

日本油脂の紹介�

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NOF CORPORATION: Our introductionInnovating Drug Delivery Systems

As an integrated chemical manufacturer featuring diverse activities from “Biosphere to Outer Space”,NOF CORP. has now focused on the development of innovative drug modifiers, carriers and solubilizerssuch as PEG, phospholipids, highly purified oleic acid nonionic surfactants, cholesterol pullulan,MPC polymers and chitosan. As a worldwide leader in drug delivery systems (DDS), NOF providesexcellent product quality for the pharmaceutical formulations field.

革新的DDSへの挑戦“バイオから宇宙まで”のキャッチフレーズの基に幅広い分野で社会に貢献する当社が、世界に誇るPEG、リン脂質、高純度オレイン酸系非イオン活性剤、コレステロールプルラン、及びMPCポリマーを駆使し、世界の医薬品関連分野に向けてDDS事業開発部を発足させました。DDSにおける世界のリーダーとして、当社はGMPグレードの優れた商品を提供しています。

PRODUCT OVERVIEWI. Activated PEGs and PEGylation technology

We produce excellent purity methoxypolyethylene glycols (mPEGs) ranging from 2,000 to 30,000molecular weight and also activated PEG derivatives including methoxy-PEG amines, maleimidesand carboxylic acids. By offering various PEG terminal groups, many customers’ drugs can be modi-fied. In addition, drug circulating half-life and biodistribution in the blood stream can be prolongedwhen our PEG-modified phospholipids are used and injected as liposomal formulations.

分子量2,000から30,000までの極めて高純度のメトキシポリエチレングリコールを製造できるだけでなく、これらの原料を用いたメトキシPEGアミン、マレイミド、カルボン酸などの活性化PEG誘導体も高純度、高品質で製造しています。PEGの末端基を変化させることにより、お客様の医薬品を様々に修飾することができます。また、当社の高純度リン脂質と高純度PEGを融合させたPEG化リン脂質を用いますと、医薬品の血中半減期を延長することができます。

II. Phospholipids and Liposome formulation technology

We have successfully supplied our quality GMP-grade phospholipids worldwide for drug productsfor decades with solid reliability and outstanding reputation. We not only develop new types ofphospholipids, but also formulate lyophilized liposomes, so-called EMPTY LIPOSOMES, with whichcustomers readily encapsulate their drugs with their methods.Our LIPONIZER liposome production systems allow reliable batch processing of customer formula-tions into liposomes. (LIPONIZER is registered mark of Nomura Micro Science Co., Ltd.)

1990年代初期からGMPグレードのリン脂質を世界に向けて供給し、市販のリポソーム医薬品に用いられ高い評価を得ています。また、顧客サイドで、その医薬品を容易にリポソーム化できる中空リポソームを開発しました。リポナイザー®を使用すればバッチ毎のバラツキの無いリポソームが作製出来ます｡

III. High-purity Oleic Acid Derivatives

Hiht-purity Oleic Acid Derivatives (NOFABLE® Series) derived from highly purified oleic acid havebeen developed so that customers can dissolve and formulate their sensitive drugs while suppress-ing degradation due to oxidation.

独自の高純度に精製したオレイン酸を用いて、誘導体の非イオン活性剤（NOFABLEシリーズ）を開発しました。酸化に由来する不純物を除去していますので、医薬品の分解や変性などが防げます。さらに、NOFABLEシリーズを用いてワクチン用のオイルアジュバントも開発しています。

IV. Low molecular weight MACROGOL (PEG200, 300, and 400)

NOF has developed high quality polyethylene glycols specific for pharmaceutical excipient use. OurSUNBRIGHT® DKH series is specifically synthesized and purified to reduce ethylene glycol and dieth-ylene glycol contaminants found in competitors’ products.

当社は、EG(エチレングリコール)およびDEG(ジエチレングリコール)の極めて少ない医薬品添加剤用ポリエチレングリコールを開発しました。

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V. Cholesterol pullulan

Pullulan partially modified with cholesterol have been developed to enable fabrication of highlypurified nanoparticles by dispersing into water. Antigens, peptides and proteins can be encapsulatedinto nanoparticles, and these conjugates can activate the immune system as adjuvant-like excipientsin vaccine or immuno-stimulating products.

多糖のプルランをコレステロールで修飾し部分的に疎水化させた後、水に均一に分散させることにより高純度のナノ粒子を作製する技術を開発しました。抗原やタンパク質をナノ粒子中に包埋でき、得られた複合体は生体の免疫細胞を活性化させます。

VI. MPC polymers and solubilizers for poorly water-soluble drugs

Unique water-soluble phosphorylcholine-containing MPC polymers in which lipid-likephosphorylcholine groups copolymerized with alkyl hydrophobic units have been developed. Usingour MPC polymers, poorly water-soluble drugs can be dissolved in aqueous media.

親水性のホスホリルコリン基を疎水系のメタクリル酸ポリマーに導入することにより、水溶性のMPCポリマーを開発しました。このポリマーを使用して、難水溶性薬物を可溶化することができます。

VII. Hydrophobic drug solubilization kits

We have developed new solubilization kits for poorly water-soluble drugs using our proprietary so-phisticated drug delivery technologies. Drug solubilizing procedures are easy so that the kit is suit-able for NCE screening or toxicological testing.

種々のDDS技術を結集し、難水溶性薬物の可溶化キットを開発しました。キットの使用方法は極めて簡単で、安全性試験、新規開発薬剤の初期テストのスクリーニングを容易に行うことができます。

VIII. Chitosan with low endotoxin contamination

Highly purified chitosan is now introduced for gene delivery or hydrogel use. Using sophisticatedpurification methods, endotoxin-free chitosan (less than 100EU/g) is available for the DDS field.

高純度のキトサンを遺伝子導入用およびハイドロジェル用途にご紹介いたします。独自の精製技術によって開発されたエンドトキシンフリーのキトサンはＤＤＳ分野に安心して御使用いただけます。

IX. Biocompatible anchors for membrane incorporation (BAM)

We have developed our BAM concept for modifying cell membranes. Using BAM, cell or liposomalmembranes can be modified with proteins or drugs without causing damage. Our new BAM technol-ogy allows live cell immobilization on surfaces of various kinds of materials.

細胞にダメージを与えない細胞修飾剤を開発しました。BAM を用いることにより細胞や組織にダメージを与えることなく細胞や組織の表面を蛋白質や薬剤などの生理活性物質で修飾することや細胞や組織を生きたまま各種材料表面に固定化することができます。

Using these high-quality products, NOF CORPORATION provides custom formulation expertise andassistance on request.

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I. High Quality PEGs and Activated PEGs <SUNBRIGHT® Series>

Drugs, enzymes, phospholipids and other biomaterials can be covalently conjugated with our acti-vated PEGs by various terminal coupling groups as shown above, facilitating versatile drug formula-tion.

この図に示しますように、末端に種々の官能基をもつPEG修飾剤を用いることにより、薬剤、酵素、リン脂質、その他生体物質にPEG鎖を導入することが可能です。また疎水性の高分子等とPEG修飾剤を結合させることにより、高分子ミセル体を形成し、疎水性薬剤等を水溶液中に安定に分散させることができます。さらに、リン脂質と結合させたリン脂質ーPEGをリポソームに用いることにより、リポソーム表面に水和層を付加することができ、薬剤等を内包したリポソーム製剤を安定に分散させることができます。

Our activated PEGs in the SUNBRIGHT series can be used for tethering to or masking colloidal orvesicle surfaces for targeting (left) or in polymeric self-assembled micellar vesicles as a hydrophiliccorona right).

以上のように、PEG修飾をすることにより、薬剤等の安定化、抗原性の低下、投与量の低減等を図ることができ、さらに抗体等を表面に結合させることによりターゲッティング性を高めることができます。

NOF has developed the following superior quality activated PEG (SUNBRIGHT Series) well-suited forpreparing pharmaceuticals and biomedical products. Our SUNBRIGHT Series comprises extraordi-narily purified terminally functional polyalkylene glycols, the most appropriate and versatile DDSformulation materials for PEG-modified drugs.

医薬品および蛋白質などの生理活性物質の調製に最適な高品質のポリエチレングリコール(PEG)修飾剤ーSUNBRIGHT Series－を開発しました。SUNBRIGHTシリーズは、極めて高純度で多様な官能基をもつPEGであり、薬剤のPEG修飾に最も適したDDS基剤です。

Polyethylene glycol chain

Various DrugProtein, Peptide, Phospholipid(Enzyme, Cytokinase etc.)

Binding siteAmide, ester, Maleimide-S etc.

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Product Classification

1. High-purity single-armPEGs

2. High-purity multi-armPEGs

3. High-purity activatedPEGs

3-1 High-purityMonofunctionalactivated PEGs

3-2 High-puritymulti-armactivated PEGs

4. Branched activatedPEGs

5. Heterofunctionalactivated PEGs

6. Comb-shaped PEGpolymers(carboxylic anhydride type)

Description

High-purity Methoxy-PEG in the SUNBRIGHT® MEH Series contains the lowestlevels of diol PEG, with the narrowest polydispersity in the world.不純物がなくかつ分子量分布もシャープなSUNBR IGHT MEHシリーズの高純度MethoxyPEGはジオールPEG含量が極めて少なく、分子量分布もシャープです。

We manufacture a variety of multi-arm PEG derivatives (2-arm, 3-arm, 4-arm and 8-arm PEGs), which are used as raw materials of multi-arm activated PEGs.当社は、2-アーム、3-アーム、4-アーム、8-アームなど種々のマルチアーム PEG誘導体製品を製造致しております。

Mono-functional SUNBRIGHT Series contains very low levels of bi-functional PEG impu-rities, so that crosslinking of drugs during PEG modification is completely suppressed.SUNBRIGHTシリーズの一官能活性化タイプは、高純度のメトキシ PEGを原料として用いていますので不純物であるジオール由来の2官能PEGをほとんど含んでいません。その為PEG修飾時に問題となる薬物の架橋反応を防止できます。

We provide next-generation multi-arm (2,3,4 and 8 arm) PEG derivatives with termi-nals activated by various functional groups.当社は、上記に示しました2-アーム、3-アーム、4-アーム、8-アームなど種々のマルチアームPEGの末端に種々の官能性基を付与した誘導体製品を供給致しております。multi-arm PEGは、ハイドロゲルの原料だけでなく、難溶性薬剤に応用することにより溶解性の向上が計れます。

Our BRANCHED PEG is a cutting-edge reagent for your new sustained-release PEGylateddrugs. In addition, NOF has many patents and know-how for utilizing our new BRANCHEDPEGs for your applications.新しい2鎖型及び3鎖型PEG誘導体の開発をおこないました。末端官能基がマレイミド基、アルデヒド基、アミノ基、NHS活性化のものにつきまして供給をいたします。

Our new HETERO-FUNCTIONAL PEG DERIVATIVES have different functional groups in-troduced to each PEG terminal. These include reactive esters(NHS), aldehydes, hy-droxy, carboxylic acids.当社は、両末端に異なる反応性基を付与したヘテロ活性化PEG誘導体を供給いたします。

These alternating copolymers introduce many reactive anhydride groups and PEG chainsinto a single polymeric molecule (Comb-shaped polymers).このシリーズは、酸無水物基とポリアルキレングリコール鎖をもつ櫛形構造の新しいタイプのポリマーです。

Summary of NOF PEG Products

1. High-purity single-arm PEGsNOF manufactures various molecular weights of high-purity mono-functionalized methoxy-PEG(mPEG) with narrow polydispersity. These high-purity mPEGs have already been commercializedinto known PEG drugs such as PEG-INTERFERON and PEG-GCSF, and other PEG-DRUGs.

不純物がなくかつ分子量分布もシャープな高純度メトキシPEGを製造しています。これらの高純度メトキシPEGは、これまでにPEG-インターフェロンPEG-GCSFなどの種々のPEG修飾医薬品に使われています。

Methoxy-polyethyleneglycol with low-diol content & narrow polydispersity

CH3O(CH2CH2O)n-H

7

(%)100

80

60

40

20

0101 102 103 104 105 106 MW

（％）�100

80

60

40

20

0101 102 103 104 105 106 MW

Comparisons of GPC chart of mPEG (MW5,000)between NOF product and competitor’s.

CompetitorNOF

NOF’s mPEGs have the lowest levels of contaminating diol content in the world. HPLC data in Figure1 clearly show our superiority in comparison to other mPEG-5000 products. Importantly, the polydis-persity of our mPEGs is also the narrowest in the world. Our other activated PEG derivatives pro-duced using our SUNBRIGHT MEH Series achieve excellent drug modification with high-purity andhigh-activity. NOF manufactures these exclusive high-purity mPEGs with molecular weights from2,000 to 30,000.

メトキシPEG、SUNBRIGHT® MEHシリーズは、他社品に比べてジオール含量が極めて少ないことが特徴です。下図に示しますように、当社のメトキシPEG-5000は、他社品に比べて不純物であるジオール含量が極めて少なく、分子量分布がシャープであることが特徴です。当社のすべてのPEG誘導体は、このMEHシリーズを原料PEGとして用いていますので、修飾剤として高純度で高活性を示します。当社は、分子量2000から30000までの高純度メトキシPEGを提供しています。

2. High-purity Multi-Arm PEGs (2-arm, 3-arm, 4-arm and 8-arm type PEGs)We manufacture a variety of multi-arm PEG derivatives (2-arm, 3-arm, 4-arm and 8-arm PEGs). Perour tradition, these derivatives are supplied with low impurities and narrow polydispersity.

当社は、2-アーム、3-アーム、4-アーム、8-アームタイプの種々のマルチアームPEG誘導体製品を製造致しております。これらのマルチアームPEGは、マルチアーム活性化PEG誘導体の原料として使用されます。

NOF CORPORATION is the sole worldwide patent holder of these high-purity mPEGs all their deriva-tives.

高純度メトキシポリエチエレングリコール及びその誘導体につきましては、各国での特許を取得しております。※Patent No. ： JP3050228, U.S.Patent 6455639, KR0358276

H-(OCH2CH2)n-OH

2-arm type DKH Series(Polyethylene glycol)

4-arm type PTE Series(Pentaerythritol, tetra-polyethylene glycol ether)

8-arm type HGEO Series(Hexa-glycerine, octa-polyethylene glycol ether)

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3. High-purity functional Acivated PEGsOur mono-functional SUNBRIGHT® Series contains very low levels of bi-functional PEG impurities, so that un-desirable crosslinking of drugs during PEG modification is completely suppressed. Also, NOF can adjust themolecular weight and supply multi-functional PEG derivatives (bi-functional, 3-functional, 4-functional, 8-func-tional and hetero-functional) according to customer needs. NOF plans and produces these derivatives fromcarefully selected raw materials. Moreover, we can make other functional or custom polyalkylene glycol deriva-tives such as PPG, PBG, PPG-PEG block or random polymers for your new drug modifications.

また当社はPEG誘導体をベースから合成設計していますので、ご要望に応じて、分子量の変更や多官能性タイプ、ヘテロタイプ等の合成など種々の対応をすることができます。さらにその他PPG(PolyPropylene Glycol)、PBG(PolyButylene Glycol)、PPG-PEGブロック／ランダムポリマー等の他のポリアルキレングリコール誘導体を用いた活性化ポリアルキレングリコールも製造することが可能です。

GPC chromatograms comparing functional PEGderivative purity, showing NOF superior quality(NHS-activated carboxyl-PEGs: M-PEG-X-NHS)

Application Chart for PEG Derivatives

当社は種々の官能基を持つPEG誘導体を取り揃えております。

Product name

CS series

GS series

AS series

HS series

NP series

AL series

PA series

DE series

SH series

MA series

Reactive group

-CO-CH2CH2-COO-NHS

-CO-CH2CH2CH2-COO-NHS

-CH2-COO-NHS

-CH2CH2CH2CH2CH2-COO-NHS

-CO2-phenyl-NO2

-CH2CH2-CHO

-CH2CH2CH2NH2

-CH2CH2CH(OC2H5)2

-CH2CH2SH

-CH2CH2CH2NHCOCH2CH2-Maleimide

Target groups

-NH2,-OH,-SH

-NH2,-OH,-SH

-NH2,-OH,-SH

-NH2,-OH,-SH

-NH2

-NH2

-COOH

-NH2

-SH,-Maleimide,-COOH

-SH

100

80

60

40

20

01.00 2.0 3.0 4.0 5.0 6.00LogM

100

80

60

40

20

01.00 2.0 3.0 4.0 5.0 6.00LogM

Competitor’sME-050CS

Coupling of PEG derivatives to Proteins: example conditions

上記の種々の官能基を持つPEG誘導体を用いた蛋白修飾の代表例を以下に示します。1.Coupling of PEG-NHS derivatives to protein amines (PEG-NHS+ Protein-NH2)

Condition 1: 50mM Phosphate buffer (pH7.2), 4C, 6hrs*1) Condition 2: Borate-phosphate buffer (pH8.0), 25C, 2hrs*2)2. Coupling of PEG-Aldehyde derivatives to protein amines (PEG-Ald+ Protein-NH2)

Condition : Sodium cyanoborohydride (10eq.), 4C, 20hrs*3)3.Coupling of PEG-Maleimide derivatives to protein cysteine (PEG-Maleimide+ Protein-SH)

Condition : 100mM Phosphate buffer (pH6.5), 4C, 4hrs4. Coupling of PEG-NH2 derivatives to protein carboxylates (PEG-NH2+ Protein-COOH)

Condition : 50mM Phosphate buffer (pH7.2),WSC(2eq), 4C, 10hrs5. Coupling of PEG-p-Nitrophenylcarbonate derivatives to protein amines (PEG-NP+ Protein-NH2)

Condition : Borate-phosphate buffer (pH8.0-8.3), r.t, overnight*2)References: *1) Abuchowski, A., et al. (1984) Cancer Biochem. Biophys. 7,175 *2) Sartore, L., et al. (1991) Appl.Biochem.Biotechnol. 27,45 *3) U.S.Patent 5,824,784

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PEG Conjugation Capabilities

PEG conjugation is known to help enhance drug performance by optimizing pharmacokinetics, in-creasing bioavailability, decreasing immunogenicity and decreasing administration frequency withstable PEG linkages. In addition, NOF can provide various linkage chemistries in our SUNBRIGHTSeries to reliably link with your chosen candidate drug molecule site-specifically. Figure 2 showsreliable clean PEG-aldehyde conjugation with human insulin across several PEG molecular weights.

l

lane M　� lane 3lane　�1 lane　�4　�

lane　�2　� ane　�5　�

molecular weight markersinsulinPEG(ME-050AL)-insulin eluate

PEG(ME-100AL)-insulin eluatePEG(ME-200AL)-insulin eluatePEG(ME-300AL)-insulin eluate

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Product name

SUNBRIGHT MENP-20H

SUNBRIGHT MENP-50H

SUNBRIGHT MENP-10T

SUNBRIGHT MENP-20T

SUNBRIGHT MENP-30T

MW

2,000

5,000

10,000

20,000

30,000

2) SUNBRIGHT® NP Series (p-Nitrophenyl-PEGs)

CH3O(CH2CH2O)nCO

O

NO2

3-1. High-purity Monofunctional Activated PEGs

1) SUNBRIGHT® CS, GC, AS, and HS Series (Carboxylated-mPEGs; NHS active esters)

NOF provides four types of carboxyl group linkages in PEG-(X)-NHS. Customers can choose appro-priate products according to their needs and the desired stability of the PEGylated drug.

カルボキシル基の結合形式(ｘ)が異なる4タイプのNHS活性化エステル-PEGをそろえています。従って目的に応じて4タイプの中から適切なものをお選び頂けます。

Product name

SUNBRIGHT ME-020CS

SUNBRIGHT ME-050CS

SUNBRIGHT ME-120CS

SUNBRIGHT ME-200CS

SUNBRIGHT MEGC-20HS

SUNBRIGHT MEGC-50HS

SUNBRIGHT MEGC-10TS

SUNBRIGHT MEGC-20TS

SUNBRIGHT MEGC-30TS

SUNBRIGHT ME-020AS

SUNBRIGHT ME-050AS

SUNBRIGHT ME-050HS

SUNBRIGHT ME-100HS

SUNBRIGHT ME-200HS

X

succinate -CO-CH2CH2-COO-

glutarate -CO-CH2CH2CH2-COO-

carboxymethyl -CH2-COO-

carboxypentyl -(CH2)5-COO-

MW

2,000

5,000

12,000

20,000

2,000

5,000

10,000

20,000

30,000

2,000

5,000

5,000

10,000

20,000

CH3O(CH2CH2O)n-X-NHS

O

O

NNHS:

SUNBRIGHT®

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Product name

SUNBRIGHT MEPA-20H

SUNBRIGHT MEPA-50H

SUNBRIGHT MEPA-12T

SUNBRIGHT MEPA-20T

SUNBRIGHT MEPA-30T

MW

2,000

5,000

12,000

20,000

30,000

6) SUNBRIGHT® MA Series (Maleimide-PEGs)

Product name

SUNBRIGHT ME-020MA

SUNBRIGHT ME-050MA

SUNBRIGHT ME-120MA

SUNBRIGHT ME-200MA

SUNBRIGHT ME-300MA

MW

2,000

5,000

12,000

20,000

30,000

CH3O(CH2CH2O)n-(CH2)3NHCO(CH2)2 N

O

O

5) SUNBRIGHT® SH Series (Thiol-PEGs)

CH3O(CH2CH2O)n-CH2CH2SH

Product name

SUNBRIGHT ME-020SH

SUNBRIGHT ME-050SH

SUNBRIGHT ME-100SH

SUNBRIGHT ME-200SH

SUNBRIGHT ME-300SH

MW

2,000

5,000

10,000

20,000

30,000

SUNBRIGHT®

Product name

SUNBRIGHT ME-050AL

SUNBRIGHT ME-100AL

SUNBRIGHT ME-200AL

SUNBRIGHT ME-300AL

MW

5,000

10,000

20,000

30,000

3) SUNBRIGHT® AL Series (Aldehyde-PEGs)

NOF owns rights to state-of-the-art synthetic methods for making amine-reactive mPEG-aldehydesthat avoid the formation of common bifunctional impurities.

当社は最新の技術を駆使し不純物である2官能性-PEGを含まないM-PEG-アルデヒドを製造しています。

CH3O(CH2CH2O)n-CH2CH2CHO

4) SUNBRIGHT® PA Series (Amino-PEGs)

NOF offers propylamine-functionalized amino-terminal PEGs.

当社はプロピルアミンタイプのアミノ-PEGを提供しています。

CH3O(CH2CH2O)n-CH2CH2CH2NH2

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3-2. High-purity Multi-Arm Activated PEGs

X-(OCH2CH2)n-X

2-arm type Bi-Functional PEG Series

Product name

SUNBRIGHT DE-034AS*

SUNBRIGHT DE-030CS

SUNBRIGHT DE-050CS

SUNBRIGHT DE-034GS*

SUNBRIGHT DE-050GS

SUNBRIGHT DE-100GS

SUNBRIGHT DE-200GS

SUNBRIGHT DE-034HS*

SUNBRIGHT DE-050HS

SUNBRIGHT DE-100HS

SUNBRIGHT DE-200HS

SUNBRIGHT DE-100MA

SUNBRIGHT DE-200MA

SUNBRIGHT DE-010PA

SUNBRIGHT DE-020PA

SUNBRIGHT DE-034PA

SUNBRIGHT DE-050PA

SUNBRIGHT DE-100PA

SUNBRIGHT DE-200PA

SUNBRIGHT DE-300PA

SUNBRIGHT DE-050SH*

SUNBRIGHT DE-100SH*

SUNBRIGHT DE-200SH*

X

Succinimidylcarboxymethyl

-CH2-COO-NHS

Succinimidylsuccinate

-CO-CH2CH2-COO-NHS

Succinimidylglutarate

-CO-CH2CH2CH2COO-NHS

Succinimidylcarboxypentyl

-CH2CH2-CH2CH2CH2-COO-NHS

Maleimide

-NHCO-CH2CH2-Maleimide

Propylamine

-CH2CH2CH2NH2

Ethanthiol

-CH2CH2SH

MW

3,400

3,000

5,000

3,400

5,000

10,000

20,000

3,400

5,000

10,000

20,000

10,000

20,000

1,000

2,000

3,400

5,000

10,000

20,000

30,000

5,000

10,000

20,000

Arms

2

4-arm type 4-arm-Functional PEG SeriesPTE Series (Pentaerythritol, tetra-polyethylene glycol ether)

XOH2CH2C

n o

o

o X

X

n

n

oX

OH2CH2C

CH2CH2O

CH2CH2O

n

SUNBRIGHT® * : custom synthesis

13

Product name

SUNBRIGHT PTE-100CS

SUNBRIGHT PTE-100GS

SUNBRIGHT PTE-200GS

SUNBRIGHT PTE-100HS

SUNBRIGHT PTE-200HS

SUNBRIGHT PTE-100NP

SUNBRIGHT PTE-200NP

SUNBRIGHT PTE-200MA*

SUNBRIGHT PTE-100PA

SUNBRIGHT PTE-200PA

SUNBRIGHT PTE-100SH

SUNBRIGHT PTE-200SH

X

Succinimidylsuccinate

-CO-CH2CH2-COO-NHS

Succinimidylglutarate

-CO-CH2CH2CH2-COO-NHS

Succinimidylcarboxypentyl

-CH2CH2CH2CH2CH2-COO-NHS

p-Nitophenylcarbonate

-CO2-phenyl-NO2

Maleimide

-NHCO-CH2CH2-Maleimide

Propylamine

-CH2CH2CH2NH2

Ethanethiol

-CH2CH2SH

MW

10,000

10,000

20,000

10,000

20,000

10,000

20,000

20,000

10,000

20,000

10,000

20,000

Arms

4

8-arm type

Product name

SUNBRIGHT HGEO-200GS*

SUNBRIGHT HGEO-200NP*

X

Succinimidylglutarate

-CO-CH2CH2CH2-COO-NHS

p-Nitophenylcarbonate

-CO2-phenyl-NO2

MW

20,000

20,000

(OCH2CH2)n

HC

HC

HC

H2C

XO

O

O

O

(CH2CH2O)n

CH2

CH2

CH2

CH2

CH2

OX

X(CH2CH2O)n

O X(CH2CH2O)n

O X(CH2CH2O)n

4

8-arm-Functional PEG SeriesHGEO Series (Hexa-glycerine, octa-polyethylene glycol ether)

Arms

8

SUNBRIGHT® * : custom synthesis

* : custom synthesis

14

4. Branched Activated PEGsNOF has developed novel branched PEG products for sustained-release PEGylated drugs.NOF offers various types of functional group, such as aldehyde, maleimide, carbonate, or carboxy-late activated esters (Y), for activated branched PEGs. A range og spacers (X) for each functionalgroup is also available. NOF’s branched PEGs are our original and patented products.

当社は、下記に示しましたように新しい2鎖型PEG誘導体の開発をおこないました。それぞれのPEG鎖の分子量または官能基までのリンカー部分を調節することが出来ます。末端官能基としては、アミノ基、アルデヒド基、マレイミド基、カーボネート基、活性エステル化カルボキシル基を揃えております

X Y

Polyethyleneglycol

PolyethyleneglycolX: SpacerY: Functional group

Product name

SUNBRIGHT GL2-200MA

SUNBRIGHT GL2-400MA

SUNBRIGHT GL2-200PA

SUNBRIGHT GL2-400PA

SUNBRIGHT GL2-200GS2

SUNBRIGHT GL2-400GS2

SUNBRIGHT GL2-200HS

SUNBRIGHT GL2-400HS

SUNBRIGHT GL2-200NP*

SUNBRIGHT GL2-400NP*

SUNBRIGHT GL3-400AL2

SUNBRIGHT GL3-400MA*

SUNBRIGHT GL3-400PA*

MW

20,000

40,000

20,000

40,000

20,000

40,000

20,000

40,000

20,000

40,000

40,000

40,000

40,000

Functional end Y

Maleimide

Amine

NHS-glutaryl

NHS-carboxymethyl

Carbonate

Aldehyde

Maleimide

Amine

SUNBRIGHT® * : custom synthesis

Image of Branched PEG-drug

Branched PEG modification offers ability to custom modify bioactive agents and surfaces site-selec-tively with dual PEG chains per site as depicted above.

15

Product name

SUNBRIGHT HO-020PA

SUNBRIGHT HO-034PA

SUNBRIGHT HO-050PA

MW

2,000

3,400

5,000

HO(CH2CH2O)n-CH2CH2CH2NH2

Product name

SUNBRIGHT PA-020HC

SUNBRIGHT PA-034HC

SUNBRIGHT PA-050HC

MW

2,000

3,400

5,000

2) Amino-PEG-Carboxylic acid

HCl・H2N-CH2CH2CH2O(CH2CH2O)n-(CH2)5COOH

Product name

SUNBRIGHT BO-100HS*

MW

10,000

3) Boc-protected-Amino-PEG-Carboxyl-NHS

Boc-NHCH2CH2CH2O(CH2CH2O)n-(CH2)5COO-NHS

5. Heterofunctional PEGs

1) Hydroxy-PEG-Amine

Product name

SUNBRIGHT HO-050AL

SUNBRIGHT HO-100AL

SUNBRIGHT HO-200AL

SUNBRIGHT HO-300AL

MW

5,000

10,000

20,000

30,000

5) Hydroxy-PEG-Aldehyde

HO(CH2CH2O)n-CH2CH2CHO

* : custom synthesis

Product name

SUNBRIGHT MA-050HS*

MW

5,000

4) Maleimide-PEG-Carboxyl-NHS

Maleimide-(CH2)3CONHCH2CH2CH2O(CH2CH2O)n-(CH2)5COO-NHS

* : custom synthesis

Boc = tert-butoxycarbonyl

16

6. Comb-shaped Co-polymers (carboxylic anhydride type)These alternating copolymers are developed for introducing many reactive anhydride groups andPEG chains into a single polymeric molecule (comb-shaped polymers) as shown below.

R1 : H or CH3

R2 : CH3 or other alkyl groupAO : alkylene oxide group

When this polymer reacts with proteins and enzymes, many PEG chains can be introduced to thebiomolecule or drug as shown below.

このポリマーを酵素の修飾に用いますと、一本鎖のPEG修飾剤を用いた場合に比べて、少ない結合で酵素表面により多くのPEG鎖を導入することができます。その結果、修飾酵素の残存活性が高くなります。

R1

C

O O OCH2

(AO)n R2

CH2 CH CH

m

O

C C

Moreover, polymer hydrophilic/lipophilic balance (HLB) can be controlled by balancing propyleneoxide and ethylene oxide ratios. Selecting appropriate polymers, customer modified proteins, en-zymes or drugs can be dissolved and stabilized in organic solvents.

さらにこの化合物は、骨格内のポリアルキレングリコールのエチレンオキサイドとプロピレンオキサイドの組成比を変えることにより、親水性－親油性のバランスを調整することができます。使用する酵素に応じて最適なポリマーを選択することにより、修飾酵素を有機溶剤に溶解することも可能です。

Method of Test

Mw of AO (PEG)

DP(1)

Total Mw

AM-0530K

about 500

about 30 ~ 40

About 15,000 ~ 20,000

AM-1510K

about 1,500

about 10 ~ 15

About 15,000 ~ 20,000

AM-2090P

about 2,000

about 10 ~ 20

about 20,000 ~ 40,000

(1) Degree of Polymerization

PEGylation usingComb-shaped co-polymers

PEG chain

Comb-shaped typepolymers

Enzyme

PEGylation usinggeneric single chain PEG derivatives

Drug, Proteinor Surface

General Characteristics for PEG SUNBRIGHT AM series

17

20

40

60

80

100

0

Res

idu

al a

ctiv

ity

(%)

(week)1 2 3 4

native

3/1

2/1

1/1

0.5/1

PEG Modifier/Enzyme ratio (wt/wt)

Efficacy of Comb-type PEG Enzyme Conjugation

Stability of Modified Protease in water (at 40˚C)

Stability of modified L-Asparaginase

Residual activity and Antigenicity of Modified L-Asparaginase

Modifier Type

Native

PEG1

PEG2

AM-1510K

AM-2090P

*Modification ratio

0(0)

79(73)

57(52)

50(46)

34(31)

Residual activity %

100

0.9

11.0

45.5

85.3

Antigenicity

100

0

0

0

0

*Number of amino groups conjugated to PEG modifier.(Total number of amino groups in ASPase= 92).

References: for modified enzymes using SUNBRIGHT AM-series:(1) Kajiuchi, J., Park, J. W. (1992) J. Chem. Eng. Japan. 25, 202(2) Masunaga, T., et al. (1993) Journal of SCCJ. 27, No.3 276(3) Sasaki, H., et al. (1993) Biochem. Biophys. Res. Commun. 197, 287(4) Kodera, Y., et al. (1994) Bioconjugate Chem. 5. 283(5) Matsuo, K., et al. (1994) Adv. Bioseparation Eng. 1993, 3, 56

20

40

60

80

100

0

Act

ivit

y (relative %

)

Incubation Time (min.)AM-2090P L-asparaginase

Stability at 65℃�

0 20 40 60 80

20

40

60

80

100

0

Incubation Time (min.)AM-1510K L-asparaginase

Stability in 4.0M Urea

0 20 40 60 80

20

40

60

80

100

0

Incubation Time (min.)Native L-asparaginase

Stability at pH 4.0

0 20 40 60 80

18

II. Phospholipid derivatives and Liposome Formulations

<COATSOME® series>NOF supplies the high-purity phospholipid derivatives (COATSOME Series) suitable for lipid emul-sion and liposome formulations used for pharmaceuticals and cosmetics. Using state-of-the-art syn-thesis and purification technology, NOF has developed various kinds and grades of phospholipidsfor pharmaceuticals and cosmetics. Also, NOF offers custom-made synthesis of phospholipids. Fur-thermore, NOF provides unique research kits for liposomal drug delivery systems (DDS) with whichcustomers can easily make and study liposomal formulations. Both liposome suspensions and equip-ment for liposome fabrication are also available from NOF (see LIPONIZER®, section X).

当社は、医薬品や化粧品などの脂肪乳剤処方やリポソーム処方に適した高純度リン脂質誘導体（COATSOME シリーズ）を、世界トップレベルの最新合成技術・精製技術を用いて提供しています。医薬・化粧品用リン脂質については豊富な品種とグレードを取り揃えています。また、お客様の要望にあわせた構造のリン脂質についてもカスタム合成いたします。さらに、簡単に薬剤のリポソーム化ができる中空リポソームキットやリポソーム処方液、リポソーム製造機も販売しています。

NOF Phospholipid Product Summary

1. Phospholipids from natural origin

天然系リン脂質

1-1. Soybean Phospholipids

1-2. Egg yolk Phospholipids

1-3. Sphingomyelin

2. Synthetic phospholipids

合成系リン脂質

3. PEGylated and Functionalized

Phospholipids

4. Lipid Mixture for Tumor Targeting

5. Empty Liposome kit

6. LIPONIZER

(see Section X)

NOF purifies phospholipids from natural origin and carefully selected starting materials.

天然系リン脂質は厳選された原料を用いて、高度に精製して純度を上げたものです。

NOF uses non-GMO products from plant origin, and virus-free products from egg origin.

植物由来のものは非遺伝子組み替え、卵黄由来はウィルスフリーの原料を使用しています。

NOF provides pure products from both milk and egg origin.

原料としてミルク由来と卵黄由来の２種類を揃えています。

NOF’s synthetic phospholipids are all plant origin and not animal origin. Saturated or unsatur-

ated acyl chains, both symmetric and asymmetric types, reactive group-attached phospholipids

and PEG-phospholipids are available.

合成リン脂質は、動物性原料を使わずに合成したものです。

飽和系、不飽和系、対称型、非対称型、反応性リン脂質、PEGーリン脂質など多くの品種を取り揃えています。

PEGylated and functionalized phospholipids are available for long-circulation and targeting use.

血中滞留性向上リポソームやターゲティング用に、PEG リン脂質及び末端活性化基結合リン脂質も揃えています。

Lipid Mixture containing transferrin attached phospholipid is supplied for tumor targeting

liposome preparation.

癌細胞へのターゲティング用にトランスフェリンを結合したリン脂質を含む脂質混合物を供給します。

Customer’s water soluble drugs are easily formulated as liposomes with this kit.

簡単手軽にお手持ちの薬剤をリポソーム化することができます。

For liposome sizing, NOF provides the Liponizer™ of which small scale to commercial scale

production is available.

小スケールから工業生産まで対応できる製造機器です。

19

1. Purified phospholipids from natural origins <COATSOME® NC series>

1-1. Soybean Phospholipids

植物（大豆）由来リン脂質誘導体

NOF supplies high-purity phospholipids of high phosphatidylcholine content with less color and lessodor using carefully selected NON-GMO soybeans. Hydrogenated forms are more stable with satu-ration fatty acids and easier to handle in both powder.

遺伝子組換え原料を使用していない厳選された天然大豆を原料に用いて、ホスファチジルコリン含量の高い高純度なレシチンを提供しています。高純度品であるため、色や臭いに優れていて、水素添加によりアシル鎖の不飽和基を少なくしていますので、非常に安定性に優れ、粉末形態であるため使いやすくなっています。

Basic Structure : di-acyl-phosphatidylcholine

R1COO, R2COO : C12~C20 fatty acids

Product

Name Type

COATSOME NC-21E Hydrogenated

COATSOME NC-21 Hydrogenated

Reference data

PC purity I.V.* Appearance

98% up Max 1 White powder

90% up Max 1 White powder

*I.V.= Iodine Value

1-2. Egg yolk Phospholipids

卵黄由来リン脂質誘導体

Phospholipids from egg yolk, purified of impurities and supplied as virus-free, are characterized bysaturated acyl chain at the α-position and unsaturated acyl chains at the β–position are basic struc-ture. Therefore, these lipids can be easily dispersed in the water to perform excellent surface-activeeffects, so that they are also formulated in lipid microspheres.

ウィルスフリーの卵黄を原料として、高度精製して不純物を除去しています。卵黄リン脂質の特徴は、リン脂質中の二つのアシル基がそれぞれ飽和と不飽和と異なっている構造のリン脂質が主成分であることです。そのため、水分散性が良く界面活性能に優れています。また、リピドマイクロスフェアーの主要構成成分の一つとして、親油性薬物のキャリアーに用いられています。

NOF offers liposome formulations suitable for cosmetics or topical use of pharmaceuticals with col-orless and odorless suspensions. There include both liquid and powder forms.

当社は、高純度なリン脂質を用いた色や臭いがない化粧品や外用剤に適したリポソーム処方を提供しています。液体・粉体の両方のタイプがあります。

20

1-3. Sphingomyelin from natural sources <COATSOME® NM series>

高純度天然スフィンゴミエリン

Phospholipids are the primary constituents of biological membranes. Recently, phospholipids havebeen directly implicated in the important biological functions as well as biomembrane structure.Sphingomyelin in particular was found to be vital for formation and maintenance of lipid rafts, whereimmune responses, various kinds of signaling, and certain material transport take place. Sphingo-myelin is thus involved in expression of specific cellular functions, such as intracellular informationtransmission and maintenance of membrane structure. Sphingomyelin, with long fatty acid chainsand low phase transition temperatures (Tc), is easy to prepare into liposomes.

Highly pure sphingomyelin of either the milk or yolk origin is available from NOF.Sphingomyelin from yolk has fatty acid chains consisting of about 80% palmitic acid joined by amidebonds, while that from milk contains many kinds of fatty acids whose chains are longer than palmiticacid. These two sphingomyelin products slightly differ in physical properties, and can be selectedaccording to needs.

リン脂質は、生体膜を構成する主要な物質ですが、近年、これらの脂質が構成脂質の役割にとどまらず、膜機能に直接関与する重要な役割を担っていることがわかってきました。中でも、スフィンゴミエリンは、ラフトの形成と維持に非常に重要であることが明らかにされています。ラフトは、免疫応答を始めさまざまなシグナリングの場として、また特定の物質輸送の場として機能していて、スフィンゴミエリンが、生体膜の構造維持とともに細胞内情報伝達系への関与など細胞の機能発現にも大きく関わる物質として大変注目されています。またスフィンゴミエリンは脂肪酸鎖長が長い割に、相転移温度（Tc）が低いという特徴があり、リポソーム化しやすい基剤です。当社は注目度の高いスフィンゴミエリンを牛乳由来のものと卵黄由来のものを高度精製して提供します。卵黄由来では、アミド結合した脂肪酸鎖の約80％がパルミチン酸であるのに対し、牛乳由来では、パルミチン酸以上の長鎖の脂肪酸が幅広く含まれていて、物理的な性質が若干異なるので、用途に応じて選択できます。

Sphingomyelin Structure

Product

Name Origin

COATSOME NM-10 Egg

COATSOME NM-70 Milk

Reference data

purity Alkyl composition

98% upR1: C13

R2: C15:0, C17:0

98% upR1: C13-C24

R2: C15:0, C17:0, C21:0, C22:0, C23:0

R1COO : C12~C22 saturated fatty acidsR2COO : C12~C20 unsaturated fatty acids

Product

Name Type

COATSOME NC-50 Non-hydrogenated

COATSOME NC-11 Hydrogenated

Reference data

PC purity Appearance

95% up White to off-white solid / powder

95% up White powder

Basic Structure : di-acyl-phosphatidylcholine

21

2. Highly-purified synthetic phospholipid derivatives

NOF produces high-purity fatty acids including unsaturated fatty acids using their own technology.Diverse synthetic phospholipid derivatives of various fatty acid compositions are available from NOF.These phospholipids contain no fatty acid materials of animal origin, are produced in FDA-inspectedfacilities under cGMP, and are safe for pharmaceutical use.NOF also synthesizes new phospholipid derivatives at customer request. NOF is also highly experi-enced in DMF registration of new derivatives and supplies lipids in both small and commercial scalequantities for approved pharmaceutical products.

原料の脂肪酸を自社で高純度に精製して、各種リン脂質誘導体を製造していますので、様々な脂肪酸組成の誘導体を合成することができます。当社の合成リン脂質は、動物由来原料を使用していません。また、FDAの認可を受けたGMP管理下の工場で製造していますので、安心して医薬品に使うことが出来ます。新しいリン脂質誘導体についても受託合成致し、新規誘導体のDMF登録も数多く行っていますので、試薬ステージから臨床ステージ、さらには承認医薬品用途まで供給できます。

NOF Synthetic phospholipid derivatives

Liposomes were imaged by FEI company Ltd.,with using Cryo-TEM ,TECNAI F20TWIN Cryo.

The figure above divides the traditional phospholipids into three characteristic parts. Each of thesecan be modified and produced by NOF to yield various derivatives. Custom properties can be ob-tained with the combination of these parts for different end-goals.

合成リン脂質は大きく三つに分かれていて、これらを組み合わせることにより、いろいろな物性の誘導体を合成することが出来ます。

22

•Synthetic phospholipid derivatives: symmetric acyl chains

Symmetric-type synthetic phospholipids have two identical acyl groups (R1, R2). Natural phospho-lipids show distributions of fatty acids that constitute acyl groups, usually different fatty acids, result-ing in fluctuation of lipid purity and quality depending on the materials source. By contrast, syntheticphospholipids do not show such fluctuations. They are highly uniform and optimal for producingdrugs. Two kinds of symmetric-type synthetic phospholipids are available from NOF: saturated andunsaturated phospholipids.

対称型はリン脂質の構造中の二つのアシル基が同一の脂肪酸から構成される合成リン脂質です。天然のリン脂質の場合、アシル基の構成脂肪酸が分布を持っていて、二つのアシル基が不均一である場合が多いため、原料の産地等によって品質がばらつく場合がありますが、合成リン脂質はそのようなばらつきはありません。均一性が厳しく要求される医薬品に最適です。対称型の合成リン脂質としては飽和脂肪酸系と不飽和脂肪酸系の２種類がございます。

•Synthetic phospholipids derivatives: asymmetric acyl chains

Asymmetric-type synthetic phospholipids have two different fatty acids introduced into the indi-vidual R1 and R2 acyl positions. Introducing pure fatty acids into the a and b positions results inconsistent characteristics such as phase transition temperatures. Using the highly pure fatty acidsfrom NOF and strictly controlling the introduction of fatty acids, where the a position is saturatedand the b position is unsaturated, NOFs lipid quality and purity are unique. This yields two character-istics: (1) stability as in saturated phospholipid derivatives, and (2) ease-of-handle, like unsaturatedphospholipids.Another asymmetric type also available from NOF is a high-quality lyso phospholipid with only the aacyl chain position substituted (see below).

非対称型の合成リン脂質は、リン脂質の持つ二つのアシル基に異なる種類の脂肪酸を導入したものです。1位と2位に純粋な脂肪酸を導入することにより、相転移温度が一定なリン脂質となります。特に、当社の持つ高純度脂肪酸を原料として、1位の位置に飽和脂肪酸、2位の位置に不飽和脂肪酸を厳しくコントロールして導入したタイプは、飽和系リン脂質誘導体の安定性と不飽和系リン脂質の取り扱い易さを併せ持つ第２世代リン脂質と言えます。また、別の非対称型として1位のアシル基のみを結合させたリゾリン脂質も提供していります。

Typical chemical structure for asymmetric phospholipids

Asymmetric (saturated/unsaturated) acyl group type (POPC):

Asymmetric saturated acyl group single chain type (Lyso phospholipid type)

23

•Saturated Unsaturated acyl group types

飽和脂肪酸系リン脂質誘導体と不飽和脂肪酸系リン脂質誘導体

NOF uses no animal materials to produce phospholipid derivatives of saturated fatty acids.

当社のリン脂質誘導体に使用している飽和脂肪酸は、動物原料を一切使用しておりません。

In general, highly pure unsaturated fatty acids are difficult to produce. Thus, lipid products usuallycontain impurities and are unstable against oxidization. However, NOF produces highly pure mono-unsaturated fatty acids such as oleic or erucic acids, very stable against oxidation. Phospholipidderivatives produced using these highly pure mono-unsaturated fatty acids are also highly pure andvery stable.All unsaturated fatty acid products from NOF are produced from plant origins.

一般的に不飽和脂肪酸は精製が困難で高純度な原料は得られにくく様々な不純物が混在するといわれています。そのため不飽和脂肪酸は酸化安定性が悪いと言われています。最新技術を駆使して安定性の極めて高い高純度モノ不飽和脂肪酸を製造していて、それを用いたリン脂質誘導体は、高純度かつ酸化安定性に非常に優れています。また、原料に使用している不飽和脂肪酸は、すべて植物を原料としたものです。

2-1. Phosphatidylcholine

R1 = identical fatty acid

Saturated acyl groups: Symmetric type (DDPC, DLPC, DMPC, DPPC, DSPC)

Product

Abbreviation Name

DDPC COATSOME MC-1010 *

DLPC COATSOME MC-2020

DMPC COATSOME MC-4040

DPPC COATSOME MC-6060

DSPC COATSOME MC-8080

Reference data

PC purity Mw Character of acyl group (R1)(purity) Tc

99% up 565.7 C10:0 99% -6

99% up 621.8 C12:0 99% 0

99% up 677.9 C14:0 99% 23

99% up 734.0 C16:0 99% 42

99% up 790.2 C18:0 99% 55

* : custom synthesis

Unsaturated acyl groups: Symmetric type (DOPC, DEPC)

Product

Abbreviation Name

DOPC COATSOME MC-8181

DEPC COATSOME MC-2121AL

Reference data

PC purity Mw Character of acyl group (R1)(purity) Tc

99% up 786.1 C18:1 99% -22

99% up 898.3 C22:1 98% -17

24

R1 and R2 are different fatty acid residues

Saturated acyl groups: Asymmetric type (MPPC, MSPC, PMPC, PSPC, SMPC, SPPC)

Product

Abbreviation Name

MPPC COATSOME MC-4060*

MSPC COATSOME MC-4080*

PMPC COATSOME MC-6040*

PSPC COATSOME MC-6080*

SMPC COATSOME MC-8040*

SPPC COATSOME MC-8060*

Reference data

R1COO R2COO PC purity Mw

C14:0 C16:0 99% up 706.0

C14:0 C18:0 99% up 734.0

C16:0 C14:0 99% up 706.0

C16:0 C18:0 99% up 762.1

C18:0 C14:0 99% up 734.0

C18:0 C16:0 99% up 762.1

* : custom synthesis

Lyso lipids (M-LysoPC, P-LysoPC, S-LysoPC, HS-LysoPC)

Product

Abbreviation Name

M-LysoPC COATSOME MC-40H*

P-LysoPC COATSOME MC-60H*

S-LysoPC COATSOME MC-80H*

HS-LysoPC COATSOME MC-70H*

Reference data

R1COO PC purity Mw

C14:0 99% up 467.6

C16:0 99% up 495.6

C18:0 99% up 523.7

HS** 99% up 518.1

* : custom synthesis HS** : Hydrogenated Soybean, Palmitoyl/Stearoyl = 1/4

Product

Abbreviation Name

MOPC COATSOME MC-4081*

POPC COATSOME MC-6081

SOPC COATSOME MC-8081*

HSOPC COATSOME MC-7081*

Reference data

R1COO R2COO PC purity Mw

C14:0 C 18:1 99% up 732.0

C16:0 C 18:1 99% up 760.1

C18:0 C 18:1 99% up 788.1

HS** C 18:1 99% up 784.3

Mixed acyl groups: Asymmetric type (MOPC, POPC, SOPC, HSOPC)

* : custom synthesis HS** : Hydrogenated Soybean, Palmitoyl/Stearoyl = 1/4

2-2. Phosphatidylglycerol

R1 = identical fatty acid

X : Na or NH4

25

Saturated acyl groups: Symmetric type (DLPG, DMPG, DPPG, DSPG)

Product

Abbreviation Name

DLPG-Na COATSOME MG-2020LS

DLPG-NH4 COATSOME MG-2020LA

DMPG-Na COATSOME MG-4040LS

DMPG-NH4 COATSOME MG-4040LA

DMPG-NH4/Na COATSOME MGLM-4040

DPPG-Na COATSOME MG-6060LS

DPPG-NH4 COATSOME MG-6060LA

DSPG-Na COATSOME MG-8080LS

DSPG-NH4 COATSOME MG-8080LA

Reference data

PG purity Mw Character of acyl group (purity)

99% up 632.7 C12:0 99% up

99% up 627.8 C12:0 99% up

99% up 688.9 C14:0 99% up

99% up 683.9 C14:0 99% up

99% up 684.6 C14:0 99% up

99% up 745.0 C16:0 99% up

99% up 740.0 C16:0 99% up

99% up 801.1 C18:0 98% up

99% up 796.1 C18:0 98% up

* : custom synthesis

Unsaturated acyl groups: Symmetric type (DOPG, DEPG)

Product

Abbreviation Name

DOPG-Na COATSOME MG-8181LS*

DEPG-Na COATSOME MG-2121LS*

Reference data

PG purity Mw Character of acyl group (purity)

98% up 797.0 C 18:1 98% up

98% up 909.2 C 22:1 98% up

* : custom synthesis

Mixed acyl groups: Asymmetric type (POPG)

Product

Abbreviation Name

POPG-Na COATSOME MG-6081LS*

Reference data

R1COO R2COO PG purity Mw

C16:0 C 18:1 98% up 771.0

* : custom synthesis

2-3. Phosphatidic acid

R1 = identical fatty acid

X : Na

26

Saturated acyl groups: Symmetric type (DMPA, DPPA, DSPA)

Product

Abbreviation Name

DMPA-Na COATSOME MA-4040LS

DPPA-Na COATSOME MA-6060LS

DSPA-Na COATSOME MA-8080LS

Reference data

PA purity Mw Character of acyl group (purity)

99% up 614.8 C14:0 99% up

99% up 670.9 C16:0 99% up

99% up 727.0 C18:0 99% up

2-4. Phosphatidylethanolamine

R1 and R2 are different fatty acid residues

Saturated acyl groups: Symmetric type (DMPE, DPPE, DSPE)

Product

Abbreviation Name

DMPE COATSOME ME-4040

DPPE COATSOME ME-6060

DSPE COATSOME ME-8080

Reference data

PE purity Mw Character of acyl group (purity)

99% up 635.9 C14:0 99% up

99% up 692.0 C16:0 99% up

99% up 748.1 C18:0 99% up

Unsaturated acyl groups: Symmetric type (DOPE, DEPE)

Product

Abbreviation Name

DOPE COATSOME ME-8181

DEPE COATSOME ME-2121AL*

Reference data

PE purity Mw Character of acyl group (purity)

99% up 744.0 C18:1 99% up

99% up 856.3 C22:1 98% up

* : custom synthesis

* : custom synthesis

Mixed acyl groups: Asymmetric type (POPE)

Product

Abbreviation Name

POPE COATSOME ME-6081*

Reference data

R1COO R2COO PC purity Mw

C16:0 C 18:1 99% up 718.0

* : custom synthesis

27

2.5. Phosphatidylserine

R1 = identical fatty acid

X : Na

Saturated acyl groups: Symmetric type (DMPS, DPPS, DSPS)

Product

Abbreviation Name

DMPS-Na COATSOME MS-4040LS*

DPPS-Na COATSOME MS-6060LS*

DSPS-Na COATSOME MS-8080LS*

Reference data

PS purity Mw Character of acyl group (purity)

80% up 701.8 C14:0 99% up

80% up 758.0 C16:0 99% up

80% up 814.1 C18:0 99% up

Unsaturated acyl groups: Symmetric type (DOPS)

Product

Abbreviation Name

DOPS-Na COATSOME MS-8181LS*

Reference data

PS purity Mw Character of acyl group (purity)

97% up 810.0 C 18:1 99% up

* : custom synthesis

* : custom synthesis

28

3-1-1. PEG-phospholipids

NOF PEG-PHOSPHOLIPIDs have three types of chemistry linking PEG with the lipid polarphosphorylethanolamine group: two are amide esters in which the ester bond may be cleaved at acertain pH, the third carbamate bond is stable enough to be used for commercial PEG-modifiedliposomes (see below).

PEG－リン脂質には、PEG鎖とリン脂質の結合様式の違いにより2タイプがあります。一つはアミド結合とエステル結合からなるタイプ、であり、pHに依存して加水分解されますが、間の炭素数によってその分解速度が異なります。もう一つのカーバメート結合タイプは加水分解に対してさらに安定であり、現在市販されているPEG－リポソームに用いられています。

R1-COO : Stearoyl, Palmitoyl, Oleoyl groupsX : amide-ester : C(O)(CH2)2C(O), C(O)(CH2)3C(O) or Carbamate linkage: C(O)Mw of PEG chain: 2,000 to 12,000

Product

Abbreviation Name

SUNBRIGHT DSPE-020C

SUNBRIGHT DSPE-050C

DSPE-PEGSUNBRIGHT DSPE-020G

SUNBRIGHT DSPE-050G

SUNBRIGHT DSPE-020CN

SUNBRIGHT DSPE-050CN

Reference data

R1COO PEG chain Mw X : Linker

2000CO(CH2)2CO

5000

C18:02000

CO(CH2)3CO5000

2000CO

5000

Liposomes Lipid emulsion

SUNBRIGHT®

3. PEGylated and Functionalized Phospholipids

3-1. PEGylated lipid derivatives

NOF’s unique PEGYLATED LIPIDS can be applied for formulating liposomal drugs for prolonged circula-tion in vivo. Coating the liposome surface with polyethylene glycol extends the circulation time of liposo-mal drugs in blood by preventing capture by the body’s reticuloendothelial system.

These derivatives are used not only for liposomes, but also for emulsifiers and stabilizers of microspheresin solution. High-purity PEG-phospholipids are produced with our high-purity methoxy-PEGs (SUNBRIGHTSeries)(low-diol content) and high-purity phospholipids (COATSOME Series).Our PEG-phospholipids are obtained with very low impurity content and narrow polydispersity importantto pharmaceutical use.

PEG修飾脂質は、体内投与において血中滞留性を高める目的でリポソーム薬剤に用いることができます。ポリエチレングリコールでリポソーム表面を覆うことにより、細網内皮系への取り込みを抑え、血中での滞留時間を延長することができます。また、乳化剤および水溶液中での微粒子の安定剤としても使用することができます。

29

3-1-2. New hydrophilic phospholipid derivativesAs shown below, we can also provide new phospholipid derivatives attached to hydrophilic poly-mers such as polyglycerine and multi-arm PEGs. These have been shown to provide liposome per-formance advantages in vivo (see below).

次に示したように、水溶性ポリマーとしてポリグリセリンおよびマルチアームPEGを付与した新しいタイプのリン脂質誘導体を供給します。

X : Linker

Product

Abbreviation Name

DSPE- SUNBRIGHT

Polyglycerine DSPE-PG8G

DSPE- SUNBRIGHT

Multi-arm PEG DSPE-PTE020

DSPE- SUNBRIGHT

Comb-shaped DSPE-

PEG AM0530K

Reference data

R1COO Hydrophilic polymer

MW Structure

700

C18:0 2,000

12,000

O

CH2OH

CH2CHO H8

CHCH2 CH CH

C CCH2

O(CH2CH2O)nCH3

O

OH

O

O

k

OH2CH2Cn o

o

o H

H

n

n

oH

OH2CH2C

CH2CH2O

CH2CH2O

n

SUNBRIGHT®

†

†DXR: doxorubicin (anti cancer drug)

30

01.0

10.0

100.0

1000.0

24 48 72 96 120Time (h)

g

/mL

DX

R in

Pla

sma

DSPE-PTE020 (1.04mM)

DSPE-PG8G (2.08mM)

DSPE-AM0530K (0.52mM)

DSPE-020CN (1.04mM)

Plasma Clearance Profile for PEG-based Liposomes and Polyglyceline

name

SUNBRIGHT GS-020

SUNBRIGHT GS-050

R1-COO

stearoyl CH3(CH2)16COO

PEG Mw

2,000

5,000

b) Cholesterol-PEG derivatives

NOF has several high-purity mono-alkylated polyethylene glycols other than our quality mPEG. Ourcholesterol-modified PEGs provide a versatile alternative to mPEG. Using more than 8-carbon chainalkylated PEGs, several unique surface-active effects are observed. These products or materials canbe used not only for surface modification of LIPOSOMES, but also for peptide PEGylation by attach-ing peptide functional groups to the PEG chain ends.

メトキシポリエチレングリコール以外にも、さまざまな種類の高純度モノアルキル置換ポリエチレングリコールを開発しています。アルキル基として炭素数が8以上の炭化水素基を用いると、種々の界面活性効果が出てきます。これらの誘導体はそのままリポソームなどの表面修飾に用いることができるのみではなく、末端に種々の官能基を導入してたん白質などのPEG化にも使うことができます。

Product Reference data

SUNBRIGHT®

3-1-3. Un-charged PEG-lipid derivatives

a) Diacylglycerol-PEG

These products lack phosphorylethanolamine groups and the extra linkage is relatively more stableagainst hydrolysis. Furthermore, because of the nonionic characteristics of the backbone, these prod-ucts are suitable to use for drugs which are sensitive to ionic charges.

このシリーズは、ジアシルグリセロールにPEG鎖を直接付与しているので、フォスフォリツエタノールアミン基およびその他の結合部位を含まないため、加水分解に対して安定であり、電荷が問題となる薬剤への使用に適しています。

31

Serum clearance efficiency of new PEG-Lipid derivatives in Liposomes

without PEG-derivative

with PEG-derivative: DSPE-010C

with PEG-derivative: GM-010

with PEG-derivative: CS-050

Liposome composition : DSPC/Chol : (1/1,mol/mol)containing 6mol% PEG-derivatives. After 6 hours of circulation

RES Blood

% serum clearance

0 25 50 75 100%

Reference : K. Maruyama., et al.(1996) Bio. Pharm. Bull. 19, 10, 1347

Typical product name

SUNBRIGHT CS-010

SUNBRIGHT CS-020

SUNBRIGHT CS-050

MW

1,000

2,000

5,000

H3CCH3

CH3

CH3

CH3

O(CH2CH2O)nH

32

3-2. Functionalized phospholipid derivatives

Functionalized phospholipid derivatives with activated groups attached at either the lipid polar endor attached PEG’s terminal end are now available from NOF. Antibodies, peptides, etc may be co-valently attached to the surface of liposomes or lipid emulsions using these reactive chemistries.These result in particles for targeting or extending circulation in blood. NOF supplies various kinds ofthese high purity derivatives utilizing our own high purity PEGs, state-of-the-art synthetic technolo-gies, and pure reagents.

機能性リン脂質を用いると、脂肪乳剤やリポソームの表面に種々のペプチドや抗体などを結合させtarget性を付与したり、PEG鎖を導入して処方の血中滞留性をあげることができます。当社は原料PEGから合成しておりますので、高純度な種々の誘導体を提供いたします。

PEG-grafted lipid

Liposome

PEG corona

peptide moiety

Targeting moiety (antibody)

3-2-1. Activated phospholipids

NOF supplies both reactive maleimido and carboxylic acid group activated phospholipids suitablefor reactions with sulfhydryl groups on antibodies or amino groups of peptides, respectively.

活性化リン脂質には、抗体などのSH基と反応するマレイミド基を持つタイプと、たんぱく質などのNH2基やSH基と反応する活性化カルボキシル基を持つタイプがあります。

Maleimido (MAL) derivative for SH group reaction Activated carboxylic acid (NHS) for NH2 group reaction

CH2-OC-R1

CH2-O-P-O-CH2CH2NHCCH2CH2CH2COH

R2-CO-CH

O

O

O

O-Na+

O O

Carboxylic acid derivative for NH2 group reaction

33

3-2-2. Activated PEG phospholipids

Product

Abbreviation Name

DSPE-PEG-NH2SUNBRIGHT DSPE-020PA

SUNBRIGHT DSPE-050PA

DSPE-PEG-MALSUNBRIGHT DSPE-020MA

SUNBRIGHT DSPE-050MA

DSPE-PEG-NHS SUNBRIGHT DSPE-020GS

SUNBRIGHT®

Reference data

Target group R1COO PEG chain Mw

COOH group2000

5000

SH group C18:02000

5000

NH2 group 2000

Reference data

Target group R1COO R2COO

C16:0 C16:0

C18:0 C18:0

SH groupC16:0 C18:1

C14:0 C14:0

C16:0 C16:0

C18:1 C18:1

C18:0 C18:0

C16:0 C18:1

C14:0 C14:0

NH2 groupC16:0 C16:0

C18:1 C18:1

C16:0 C16:0

C18:0 C18:0

C18:1 C18:1

Product

Abbreviation Name

DPPE-MAL COATSOME FE-6060MA3*

DSPE-MAL COATSOME FE-8080MA3*

POPE-MAL COATSOME FE-6081MA3*

DMPE-MAL COATSOME FE-4040MA3*

DPPE-MAL COATSOME FE-6060MA3*

DOPE-MAL COATSOME FE-81812MA3*

DSPE-NHS COATSOME FE-8080SU5*

POPE-NHS COATSOME FE-6081SU5*

DMPE-NHS COATSOME FE-4040SU5*

DPPE-NHS COATSOME FE-6060SU5*

DOPE-NHS COATSOME FE-8181SU5*

DPPE-Glu COATSOME FE-6060GL*

DSPE-Glu COATSOME FE-8080GL*

DOPE-Glu COATSOME FE-8181GL*

COATSOME® * : custom synthesis

CH2-OC-R1

CH2-O-P-O-CH2CH2NHCO(CH2CH2O)nCH2CH2CH2NHCCH2CH2CH2CO-N

R1-CO-CH

O

O

O

O-Na+

O O O

Activated carboxylic acid(NHS)-teminal (DSPE-PEG-NHS)

O

O

Maleimido-terminal (DSPE-PEG-MAL)

Amine-terminal (DSPE-PEG-NH2)

34

4. Lipid Mixture for Tumor Targeting

Ordinary tumor cells are abundant with transferrin receptors. This means that liposomes with trans-ferrin attached on the outside surface can be a potential carrier of anti-cancer drugs to tumor cells.NOF has developed a tumor targeting Lipid Mixture called COATSOME PL-001T, which can be pre-pared as a liposomal suspension in the customer’s laboratory.COATSOME PL-001T consists of PC and other suitable lipids whereby transferrin is attached withactivated phospholipid through chemical bonding.The attached document describes the liposomal preparation of COATSOME PL-001T for tumor targeting.

癌細胞表面には一般的にトランスフェリンレセプターが多く存在することから、外側表面にトランスフェリンを化学結合させたリポソームを用いることにより、癌細胞へのターゲテイングが可能になります。日本油脂の提供する脂質混合物を使用すれば、癌細胞標的能を有するリポソームをラボにて調製できます。COATSOME　PL-001T　は、ホスファチジルコリンやトランスフェリンの結合したリン脂質などを含む特殊な脂質混合体であります。ご購入の際に添付される方法に従って調製頂ければ、癌細胞標的能を有するリポソームが作製できます。

Product Name

COATSOME PL-001T

Characteristics

Dried powder consists of PC and other suitable lipids containing transferrin

attached phospholipid

35

NOF has developed a unique research kit for liposomal drug delivery system (DDS) development. Gen-erally, researchers using liposome systems intended for DDS have difficulties consistently encapsulat-ing liposomal drugs principally because development of a liposomal drug requires conditions that care-fully control the formulation of phospholipids, particle size, and encapsulation efficiency.NOF has formulated and manufactured ready-to-use freeze-dried liposomes, called Empty Liposomes,in vial form. Empty Liposomes can encapsulate drug by adding a drug solution to our Empty Lipo-somes and then gentle shaking. This allows the researcher to prepare liposomal drugs without com-plicated methods (e.g., extrusion). Encapsulation efficiency for Empty Liposomes has been found tobe especially high with anthracycline and aminoglycosides.In addition, we have developed new cationic Empty Liposomes <COATSOME EL-01-D> for gene de-livery. This novel cationic liposome contains O,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14, see below), DOPE and cholesterol, and has demonstrated effi-cient transfection activity both in vitro in serum-containing medium and in vivo.

一般に、リポソーム化医薬品を作製するためには、リン脂質組成、粒径調整、内包化率などの調整が必要であり、研究者にとってリポソーム化医薬品が容易に処方できない原因となっていました。当社はこれらの問題を解決すべくリポソーム型DDS検討用にユニークな研究用キットを開発しました。中空リポソームと称するバイアルに入った凍結乾燥リポソーム粉末は、数種類のリン脂質と電荷物質で構成されていて、医薬品を溶解させた溶液をバイアルに注入し緩やかに混和することにより、簡単に医薬品をリポソーム中に内包させることができます。中空リポソームを用いることによりエクスツルージョン等の特殊な技術を用いることなく、特にアントラサイクリンやアミノクルコシドなどの医薬品を高収率で容易に内包できます。また、遺伝子デリバリー用として新しいカチオニックリポソーム<COATSOME EL-01-D>を開発しました。COATSOME EL-01-Dは、カチオン性脂質であるO,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14)に加え、DOPEとコレステロールを含有し、in vitroの血清添加培地中やin vivoアッセー系においても高い遺伝子導入・発現活性を有している新規なカチオン性リポソームです。

O,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14)

Product characteristics are shown below. Cationic charged liposomes are prepared using stearylamine as a charged lipid. Nonionic and anionic charged liposomes are prepared using DPPG ascharged lipids.When drug solutions (using distilled water for injection) are prepared, the molarity of charged lipidrequired determines the drug concentration. Please refer to Suggestions for Use in section 3-2 below.

製品の組成と特性を以下に示します｡カチオン性リポソ－ムは電荷脂質であるステアリルアミンを処方に入れて作製しています｡ノニオン性リポソ－ムとアニオン性リポソ－ムは、電荷脂質であるDPPGのモル数を変化させて作製しています｡薬物を水（注射用蒸留水）に溶解させて薬物水溶液を作製する際には、電荷脂質のモル数に応じて濃度を決める必要があります｡詳細は2項の作製法をご覧ください｡

5. Empty Liposomes <COATSOME® EL Series>

Liposomes for Basic Research

36

(1) (2) (3) (4)

Liposome Preparation is simple

(1)Remove COATSOME EL from a cool storage temperature and allow it to come to room temperature.(2)Add a drug aqueous solution in the temperature range of 16˚C to 40˚C.(3)Shake the vial gently three to five times by hand.(4)The liposomal drug is now ready for use.

（1）COATSOME ELを冷蔵庫から取り出し、室温に達するまで放置します。（2）温度16℃から40℃の範囲内で、薬物水溶液を注入します。（3）バイアルを手に持ち、3回から5回振ります。（4）リポソーム化医薬品の出来上がり。

5-1. EL Series Product Characteristics

a) COATSOME EL-01-C, EL-01-N, EL-01-A

EL-01-C

Cationic

DPPC:Cholesterol:Stearyl

amine=52:40:8

57 mg

Stearyl amine

(8µmol/vial)

100-300

0.8-1.1

EL-01-N

Nonionic

(Slightly anionic)

DPPC:Cholesterol:DPPG

=54:40:6

61 mg

DPPG

(6µmol/vial)

100-300

0.8-1.1

EL-01-A

Anionic

DPPC:Cholesterol:DPPG

=30:40:30

61 mg

DPPG

(30µmol/vial)

100-300

0.8-1.1

Product Name

Type of Electrical charge

Lipid components

(µmol/vial)

Total amount of lipids

Charged lipid

(µmol/vial)

Particle size (nm)

Osmotic pressure ratio*

*) liposome after addition of 2mL of distilled water

EL-11-C

Cationic

POPC:Cholesterol:Stearyl

amine=52:40:8

57 mg

Stearyl amine

(8µmol/vial)

50-250

0.8-1.1

EL-11-N

Nonionic

(Slightly anionic)

POPC:Cholesterol:POPG

=54:40:6

61 mg

POPG

(6µmol/vial)

50-250

0.8-1.1

EL-11-A

Anionic

POPC:Cholesterol:POPG

=30:40:30

61 mg

POPG

(30µmol/vial)

50-250

0.8-1.1

Product Name

Type of Electrical charge

Lipid components

(µmol/vial)

Total amount of lipids

Charged lipid

(µmol/vial)

Particle size (nm)

Osmotic pressure ratio*

*) liposome after addition of 2mL of distilled water

b) COATSOME EL-11-C, EL-11-N, EL-11-A

37

New COATSOME EL-11-C, EL-11-N and EL-11-A contain POPC (COATSOME MC-6081, see p24) whichis similar to EPC (egg yolk phospholipid). Our POPC synthesized from high quality plant origin mate-rials is virus-free.

COATSOME EL-11-C、EL-11-N、EL-11-Aは、卵黄リン脂質に似た性質を持つPOPC (COATSOME MC-6081、p24参照)を配合した新しい製品です。このPOPCは、高い品質の植物系原料を用いて合成されたウィルスフリーのリン脂質です。

c) COATSOME EL-01-PN, EL-01-PA (Long-circulating Liposomes)

EL-01-PN

Slightly anionic

DSPE-PG8G:DPPC:Cholesterol:DPPG

=4.2 : 20.5 : 15.2 : 2.3

29 mg

DPPG, (DSPE-PG8G)

(2.3µmol/vial), (4.2µmol/vial)

50-250

0.8-1.1

EL-01-PA

Anionic

DSPE-PG8G:DPPC:Cholesterol:DPPG

=4.2 : 11.4 : 15.2 : 11.4

29 mg

DPPG, (DSPE-PG8G)

(11.4µmol/vial), (4.2µmol/vial)

50-250

0.8-1.1

Product Name

Type of Electrical charge

Lipid components

(µmol/vial)

Total amount of lipids

Charged lipid

(µmol/vial)

Particle size (nm)

Osmotic pressure ratio*

*) liposome after addition of 2mL of distilled water

New COATSOME EL-01-PN and EL-01-PA contain DSPE-Polyglycerine (SUNBRIGHT DSPE-PG8G, seep29) which has similar effect with DSPE-PEG regarding long circulation in blood.

COATSOME EL-01-PN、EL-01-PAは、ポリグリセリン-DSPE (SUNBRIGHT DSPE-PG8G、p29参照)を配合した新しい製品です。このポリグリセリン-DSPEは、PEG-DSPEのように血中滞留性を高める効果がありますので、血中滞留型リポソームを簡単にご使用いただけます。　

d) COATSOME EL-01-D (For the delivery of DNA)

Cationic Liposomes for the delivery of DNA with a particle size of 100-200nm and osmotic pressureratio of 0.8-1.1DOPE:Cholesterol:DC-6-14=0.75:0.75:1.00 (µmol/vial) Total amount of lipids:1.51mg.Charged lipid is DC-6-14 (1.00µmol/vial) in this formulation.Instructions for encapsulation are attached to the products in this case.

COATSOME EL-01-Dについては、別途調整方法が添付されます｡

5-2. Suggestions for use

(1) For COATSOME EL-01-C, EL-01-N, EL-01-A, EL-11-C, EL-11-N, EL-11-A, EL-01-PN and EL-01-PA

a) After 2ml of distilled water for injection are added to the vial, the osmotic pressure ratio of theproduct will be 0.8 to 1.1 Adjust the osmotic pressure ratio according to the intended experiment(e.g., influence of osmotic pressure in in vivo testing).

b) To enhance the encapsulation efficiency of the drug by electrostatic interaction, drug solution shouldbe added according to amounts of the charged lipids representing stearyl amine, DPPG respectively.The charged lipid/drug molar ratio should be no less than 2 and preferably no less than 3.

c) The aqueous solution of drug must be kept in the temperature range of 16˚C to 40˚C.d) The prepared drug encapsulated in the liposomes should be used immediately after preparation.

(Not for human use)

使用方法使用方法使用方法使用方法使用方法（a）2mlの蒸留水を添加することにより浸透圧が0.8-1.1となるように調整してあります。目的の実験によっては浸透圧の調整が必要です。（b）電気的相互作用により内包率を向上させるためには、電荷脂質(ステアリルアミン､DPPGのいずれかが該当)の医薬品溶液に対するモル比が重要であり、そのモル比は2以上で好ましくは3以上です。

（C）医薬品の水溶液は16℃から40℃の範囲に調整しておきます。（d）調整したリポソーム化医薬品は直ちにご使用ください。（人には使用できません）

38

5-3. Analysis of encapsulation efficiency

1) Method to remove unencapsulated drugs

a) DialysisPrepared suspension of drug liposomes is placed into a dialyzing tube and dialyzed against anisotonic solution. Drug not encapsulated by liposomes is discharged into the isotonic solutionoutside the tube.

b) Gel FiltrationPrepared suspension of drug in liposomes is passed through a column filled with a gel filtrationcarrier under isotonic saline (150mM NaCl). Liposomes will pass through first and unencapsulateddrugs will elute later. Select gel filtration (e.g. Sephadex G-50 for low molecular weight, Sepharoce4B for high molecular weight) media according to the molecular weight of the drug to be removed.

c) CentrifugationCentrifugation is carried out by high rotation (100,000 rpm) with isotonic saline solution (150 mMNaCl). Liposomal drug will settle to the bottom. Unencapsulated drug remains in the supernatant.Repeat the procedure two or three times at 100,000 rpm.

In order to obtain good results it is extremely important to equalize the osmotic pressure both insideand outside the liposomes. Temperature must be kept below liposome phase transition temperatureduring the entire procedure (below 40˚C).

内包化率の測定内包化率の測定内包化率の測定内包化率の測定内包化率の測定（1）未内包化医薬品の除去による方法a）透　析調整したリポソーム液はを透析チューブに入れて、等張液により透析します。未内包医薬品は等張液中に漏出します。

b）ゲルろ過調整したリポソーム液は、ゲルろ過用充填剤を入れたカラム中を通過させます。リポソームが先に、未内包医薬品が後からカラムを通過して出てきます。医薬品の分子量によって、充填剤の種類（例:Sephadex G-50:低分子用、Sepharoce 4B:高分子用）を選択します。

c）遠心分離生理食塩水（150mM NaCl）を加えて高速度下で遠心分離を行うと、リポソーム化医薬品は沈んで得られます。上澄みを除去した後再度生理食塩水を加え、通常は100,000回転で2回から3回繰り返します。これらのどの方法においても、リポソームの内層と外層を等張に維持することが必須であり、調整工程を通じて温度はリン脂質の相転移温度以下に保たなければなりません。この場合は40℃以下で行います。

2) Quantification of encapsulated drug

After separating and removing nonencapsulated drug in liposomes according to a method men-tioned above in (1), drug encapsulated in liposomes can be quantified by destroying a sample ofliposomes by adding an appropriate quantity of surface active agent (Triton X-100) or by addingsolvent (chloroform). When 2 ml of chloroform is used, 2 ml of methanol and 1.2 ml of water are alsoadded into the tube. Then vortex, followed by centrifugation for 5 minutes under 3000 rpm. Afterdestroying the liposomes by one of these methods, released drugs are quantified by ordinary meth-ods such as HPLC.

（2）内包医薬品の定量上述したいずれかの方法で未内包医薬品を除去した後、リポソームを破壊する方法によってリポソーム中に内包された医薬品の定量を行います。リポソームを破壊するには、界面活性剤を添加する方法と溶剤（クロロホルム）を入れて水相と溶剤相とに分離する方法があります。いずれにしても、リポソームを破壊した後は、通常の方法にて医薬品の定量を行います。

39

Drug

Bucladesine sodium (0.5mg/ml)

Sodium salicylate (0.16mg/ml)

DNA (Salmon Testes, 100ug/ml)

Particle Size (nm)

173

162

942

Encapsulation Efficiency (%)

20-25

40-45

100

5-4. Application Data for the COATSOME® EL Series

(1) Cationic Liposomes, COATSOME EL-01-C

(2) Nonionic Liposomes, COATSOME EL-01-N

If higher encapsulation efficiency is desired, the concentration of cationic drug must be below 1 mM.The DPPG (3mM)/drug molar ratio must be higher than 3 in order to obtain high encapsulation effi-ciency.

内包効率を向上させるためには、カチオニック医薬品の濃度は1mM以下とする必要があります。高い内包効率を得るためには、DPPG（3mM）と医薬品とのモル比は3以上とする必要があります。

(3) Anionic Liposomes, COATSOME EL-01-A

Drug Maker

Mannitol

Inulin

Liposome

EL Series

Bangham Method

EL Series

Bangham Method

AUC(%dose/ml-min)

237

2204

2137

2315

MRT(min)

448

398

408

373

CL tot(ml/min/Kg)

0.174

0.179

0.175

0.174

Drug

Doxorubicin hydrochloride (1mg/ml)

Amikacin sulfate (1mg/ml)

Streptomycin sulfate (1mg/ml)

Procainamide hydrochloride (1mg/ml)

Epirubicin hydrochloride (1mg/ml)

Pirarubicin hydrochloride (1mg/ml)

Particle Size (nm)

150

140

145

152

146

129

Encapsulation Efficiency (%)

97-100

98-100

95-100

80- 85

97-100

97-100

If higher encapsulation efficiency is desired, the concentration of cationic drug must be below 5 mM.The DPPG (15mM)/drug molar ratio must be higher than 3 in order to obtain high encapsulation effi-ciency.

内包効率を向上させるためには、カチオニック医薬品の濃度は5mM以下とする必要があります。高い内包効率を得るためは、DPPG（15mM）と医薬品とのモル比は3以上とする必要があります。

Comparison of activity in vivo*

*K.Yachi et al, Biopharm. & Drug Dispos., 17, 589-605(1996):Composition:DPPC:DPPG:Chol=27:53:20 (Anionic Liposomes)

(4) Cationic Liposomes for gene delivery (COATSOME EL-01-D)

Transfection Activities in vitro*1 by Luciferase Assay*5 (Light units/mg protein·sec)

HRA MEIIL ES-2

Serum (-) 6,048 6,291 1,325

Serum (+) 11,378 2,913 689

40

*1) Transfection performed in the absence or presence of 10% fetal bovine serum (FBS). Data shown representmean of three experiments.

*2) mEIIL cells growing in peritoneal cavities of nude mice were transfected with liposome/CAG-lacZ (20µg) andthe percentage of lacZ-positive cells was determined. Data shown represent mean:± SD.

*3) Percentages of LacZ-positive cells and labeling indexes were determined. Transfection was performed in thepresence of 10% FBS. Data shown represent mean ± SD. n=3

*4) ND = Not done*5) Reference: A. Kikuchi et al., HUMAN GENE THERAPY, 10: 947-955 (1999).

Percentage of LacZ-positive cells n

COATSOME EL-01-D 1.00 ± 0.11 5

Commercially available A 0.38 ± 0.26 3

Commercially available B 0.62 ± 0.21 3

Commercially available C 0.23 ± 0.23 3

Cell lines Percentage of LacZ-positive cells Labeling index (%)

COATSOME EL-01-D Commercially available C

Cancer cell lines

HRA 42.9± 3.8 4.7 ± 1.0 68.2 ± 1.6

mEIIL 4.5± 1.0 0.6 ± 0.1 41.0 ± 1.2

ES-2 23.7± 1.9 3.3 ± 0.9 41.9 ± 4.5

OVHS-1 0.8± 0.4 0.9 ± 0.3 6.1 ± 1.4

MCAS 1.3± 0.1 < 0.1 51.5 ± 6.1

SKOV3 11.6± 1.8 0.2 ± 0.2 35.2 ± 6.7

OVCAR3 12.3 ± 1.1 0.4 ± 0.4 34.4 ± 3.7

KK 2.7 ± 0.7 0.1 ± 0.1 26.2 ± 4.3

KOC-3S 5.9 ± 2.1 2.6 ± 1.6 ND*4

Nakajima 4.9 ± 1.7 0.1 ± 0.1 ND*4

KF 15.5 ± 2.3 0.1 ± 0.1 ND*4

SW626 26.8 ± 4.6 0.2 ± 0.1 ND*4

Colo320DM 32.3 ± 3.2 5.2 ± 1.1 ND*4

HRA 42.9 ± 3.8 4.7 ± 1.0 ND*4

mEIIL 4.5 ± 1.0 0.6 ± 0.1 ND*4

Normal cell lines

Fibroblast (TIG) 9.6 ± 2.2 0.9 ± 0.2 42.4 ± 10.7

Fibroblast (IMR) 2.5 ± 0.4 1.6 ± 0.5 14.2 ± 3.2

HUVEC0103 < 0.1 < 0.1 22.2 ± 1.6

HUVEC1204 < 0.1 0.2 ± 0.1 11.3 ± 0.2

HUVEC0923 < 0.1 < 0.1 18.2 ± 4.1

Transfection Activities in vivo*2 in intraperitoneal disseminated tumors*5

Relationship Between Transfection Efficiencies and Cell Mitotic Activities*3,*5

41

III. High-purity Polysorbate 80

NOFABLE ESO-9920 of NOF Corporation is an extra highly purified Polysorbate 80 and an extremelylow impurity profile based on both of a purification technology of Oleic Acid (99% up) and a nobleethoxylation technology.NOFABLE ESO-9920 provides a low allergic reaction among Polysorbate 80. New study indicatesthat NOFABLE ESO-9920 has a low degranulation characteristics compare with competitor’s inject-able Poylsorbate 80 due to its low impurity profile.

日本油脂の高純度オレイン酸誘導体ポリソルベート80（ＮＯＦＡＢＬＥ ＥＳＯ-９９２０）は純度が99%以上という、非常に純度の高いオレイン酸を使用しているため、脂肪酸に由来する不純物が極端に少ない製品となっています。 この特徴は安全性にもつながっており、最新のデータではラット肥満細胞を用いた試験において一般のポリソルベート80に比較してヒスタミン遊離が少ないという結果が得られていて、アレルギーを起こしにくいポリソルベートとして注目を集めています。

Characteristics of High-purity Polysorbate 80

Purity of Oleic acid (%)

Odor

NOFABLE ESO-9920

99

-

Conventional Polysorbate 80

65

+++

Polyoxyethylene Sorbitan Oleate (Polysorbate 80)

H(OH2CH2C)aO O(CH2CH2O)bHO(CH2CH2O)cH

CHCH2O-C-(CH2)7CH=CH(CH2)7CH3O

OTrade Name

NOFABLE ESO-9920

Oleic acid Content

>99%

CAS NO.

9005-65-6

Product Name

Polyoxyethylene Sorbitan monooleate

Appearance comparison of High-purity Polysorbate 80

42

Degranulation Test (An allergic study model)

Effect of Polysorbate 80 on degranulation in RBL-2H3 mast cell. The cells were treated with differentconcentration of polysorbate 80 for 60 mins. The degree of Degranulation is determined by mea-surement of released β -hexosaminidase into supernatants.

Cell Toxicity Test

Effect of concentration of Polysorbate 80 on cell toxicity using SIRC Cell. The cells were treated witheach Polysorbate 80 for 24 hrs. The viable cells are determined by Neutral Red Uptake method.

Hemolysis Test

Effect of Concentration of Polysorbate 80 on Hemolytic Ratio. Red Blood cells from guinea pig aretreated with Polysorbate 80 for 60 mins. Each hemolytic ratio is determined by absorbance of 576nmin the solution.

43

Status of European Pharmacopoeia (EP)NOFABLE ESO-9920 which consists of high purity Oleic Acid (99% up), has not met EP 5th Editioncompendium due to a cap of purity (85% max.) of Oleic Acid of Polysorbate 80 in the compendium.The cap of the purity of Oleic Acid has been eliminated in EP 5.4th Edition through negotiationsbetween European Medicals Agency (EMEA) and NOF CORPORATION. Now, NOFABLE ESO-9920 (Polysorbate 80) meets Multi-compendial-NF, EP and JP

European　Pharmacopoeia (EP) 5th EDITIONに収載されているポリソルベート80にはオレイン酸純度の上限が規定（５８～８５％）されており、ESO-9920はその高純度(99%以上)が故にEPの規格から外れるという矛盾が生じておりました。日本油脂がEP当局に働きかけた結果、EP追補5.4 にてオレイン酸純度の上限が撤廃されました。これにより、日本油脂のポリソルベート80(ESO-9920)は3極(JP、EP、NF)に対応した製品となります。

Survival Rate

Effect of concentration of Polysorbate 80 on Survival Rate in BALB/c mice. Polysorbate 80 is dilutedby PBS and injected into mice with intravinous administration.

120

100

80

60

40

20

00.5 0.75 1

Su

rviv

al R

ate

(%)

Dose (ml/mice)

44

CH3(CH2)7CH=CH(CH2)7COOHTrade Name

EXTRA OLEIN 99

Oleic acid Content

>99%

CAS NO.

112-80-1

Product Name

Oleic Acid

Formation of peroxides in oleic acid during incubation at 50℃�.

0.00 5 10 15

1.0

2.0

PO

V (

meq

/ k

g)

0.00 5 10 15

1.0

2.0

PO

V (

meq

/ k

g)

Time (hrs)

ConventionalOleic Acid

EXTRA OLEIN 99

Oxidation Stability of Oleic Acid

EXTRA OLEIN 99(High-purity Oleic Acid)

Conventional Oleic Acid(Low-Linoleic type)

Gas chromatogram for NOF Oleic acid compared to con-ventional product

Dermal Absorption Enhancement with Oleic Acid

Crystal of Oleic Acid

0

5

10

15

20

25

30

35

0 1 2 4 6 8 10

Time (hr)

Der

mal

Per

mea

tio

n (

% D

ose

)

without Oleic acid

EXTRA OLEIN 99

Conventional Oleic acid

Dermal Absorption Enhancement of Indomethacin withHighly Purified Oleic Acid (EXTRA Series).

IV. High-purity Oleic Acid Derivatives <NOFABLE® Series>

NOF has succeeded to produce 99% purity of oleic acid commercially. NOF also produces highlypurified oleic acid derivatives (glycerol-oleate, ethyl-oleate, polyoxyethylene-sorbitan-monooleate,etc.) for pharmaceutical use. These products contain reduced amounts of impurities such as perox-ides or aldehydes typically seen in oleic acid product.NOFABLE Series is obtained colorless and odorless products. Our NOFABLE ESO-9920 (polysorbate80) is highly recommended as a nonionic emulsifier (injection grade) for drug products becausedegradation of drugs by oxidation is suppressed.

99%のオレイン酸を工業的なプロセスで生産する方法を開発しました。酸化され易い不飽和脂肪酸を除去しているので、酸化に対する安定性も向上し、過酸化物やアルデヒド含量の少ない誘導体が得られ、医薬品用途には最適です。NOFABLEシリーズは写真にも示したようにほとんど無色透明かつ無臭に近い製品です。特に、NOFABLE ESO-9920（ポリソルベート80）は、酸化物の少ない点から医薬品の分解を極力防止できる製品として、注射医薬品の非イオン系乳化剤として推奨します。

1. High-purity Oleic Acid <EXTRA OLEIN® 99>

45

2-2. Glycerol Oleate

2-3. Ethyl Oleate

HO-CH2-CH(OH)-CH2-OCO-(CH2)7CH=CH(CH2)7CH3

CH3(CH2)7CH=CH(CH2)7COOCH2CH3

Trade Name

NOFABLE GO-991

Oleic acid Content

>99%

CAS NO.

25496-72-4

Product Name

Glycerol monooleate

Trade Name

NOFABLE EO-99

Oleic acid Content

>99%

CAS NO.

111-62-6

Trade Name

NOFABLE SO-991

Oleic acid Content

>99%

CAS NO.

1338-43-8

OH

CHCH2O-C-(CH2)7CH=CH(CH2)7CH3

HO OH

O

O

Product Name

Sorbitan monooleate

*1 JP : Japanese Pharmacopeia

JPE: Japanese PharmaceuticalExcipients

EP : European Pharmacopeia

NF :Natural Formura

Regulatory status*1

JPE

JPE

JP EP NF

JPE

JPE

Product Name

Oleic acid

Sorbitan monooleate

Polyoxyethylene sorbitan monooleate

Glycerol monooleate

Ethyl oleate

2. High-purity Oleic Acid Derivatives

2-1. Sorbitan Oleate

Product Name

Ethyl oleate

46

V. High-purity low molecular weight MACROGOL

(PEG-200,300,400)SUNBRIGHT® DKH-02HB,DKH-03HB,DKH-04HB

NOF has developed high quality low molecular weight Polyethylene Glycols specific for pharmaceu-tical excipients.European Pharmacopoeia (EP) and U.S. Pharmacopoeia (USP) both have regulations for EG (Ethyl-ene Glycol) and DEG (Diethylene Glycol) content, due to their toxicity (EP not more than 0.4%, USPnot more than 0.25%). However, it is very difficalt to achieve low levels of EG and DEG contaminantsin low molecular weight PEGs.Our SUNBRIGHT DKH series has a much reduced EG, DEG content than competitors, so NOF canguarantee more strict specifications than EP, USP regulations. Moreover, we have eliminated othercommon impurities, such as ethylene oxide, dioxane, peroxide and aldehyde derivatives.Our specifications are shown below. NOF can also supply PEGs that meet JP, EP and USP specifications.

EG(エチレングリコール)およびDEG(ジエチレングリコール)の極めて少ない医薬品添加剤用ポリエチレングリコールを開発しました。毒性の点からEGとDEGの含有量をEPは4000ppm以下、USPは2500ppm以下に規制しています。当社、EP、USP規格を大きく下回るEG、DEGの含有量を保証します。さらにエチレンオキサイド、ジオキサン、過酸化物、アルデヒド誘導体などの不純物を取り除いています。EP、USP、JP、に適合したPEG200、300、400も供給できます。

1) EG, DEG content in PEG 200

EG (ppm)

DEG (ppm)

DKH-02HB (NOF)

61

181

company A

217

29,095

company B

11,494

43,548

EG, DEG content of PEG 400

EG (ppm)

DEG (ppm)

DKH-04HB (NOF)

41

31

company A

740

836

company B

630

753

2) Specifications

Completeness and color solution

Viscosity

Average molecular weight

pH

Residue on ignition (%)

Heavy metals (ppm)

Free ethylene oxide and

1,4-dioxane (ppm)

Limit of ethylene glycol and

diethylene glycol (%)

Organic volatile impurities

DKH-02HB

3.9-4.8

190-210

DKH-04HB

6.8-8.0

380-420

USP

DKH-03HB

5.4-6.4

285-315

MethodSpecification

Pass

Pass

4.5-7.5

NMT 0.1

NMT 5

NMT 10

(Ethylene oxide : NMT 1, 1,4-Dioxane : NMT 10)

NMT 0.05

3) Other specifications

Peroxide value (meq/Kg)

Formaldehyde (ppm)

NMT 3

NMT 5

NOF

NOF

NOF can also supply products without anti-oxidant agent, BHT.

Parameter

47

VI. Cholesterol Pullulan

(PUREBRIGHT® CP-100T)NOF has developed unique hydrogel-nanoparticles based upon hydrophobic polysaccharides(PUREBRIGHT CP) for DDS in which specific drugs and/or peptides can be encapsulated. Pullulanstarch partly modified with cholesterol (CP) shows unique characteristics described below.In water, attached cholesterol molecules form hydrophobic cores, then these cores cause polymerself-aggregation localizing attached pullulan outside. Resulting CP-nanoparticles can stabilize pro-teins and/or peptides by forming hybrid-complexes1) shown below. These particles also stimulatethe immune system through interactions with dendritic cells, so that vaccine therapy for cancer maybe demonstrated by encapsulating cancer-specific antigens, such as Her22).CP structure and characteristics are shown below.

薬物やタンパク質等を内包可能な、疎水化多糖(PUREBRIGHT CP)からなるユニークなハイドロゲルナノ微粒子を開発いたしました。コレステリル基を導入したプルラン（CP）は、水中でCP中のコレステリル基を非共有結合の架橋点として数分子間で自己会合することにより、単分散なナノ微粒子を形成します。さらに、このCPナノ微粒子は、下図に示すような様々な球状タンパク質と複合体を形成することから、酵素の安定化や薬物のキャリアとしての有用性が明らかとなっています 1）。また、癌抗原タンパク（Her2）を複合化することで、生体の免疫システムを活性化させる癌ワクチン療法の開発にも成功しています 2）。CPの構造と特長を以下に示します。

R = CONHC6H12NHCOO

OH

OH

O

OCH2

HO

O

OH

OH

O

CH2OR

O

OH

OH

m

CH2OH

O

OH

OH

O

OCH2

HO

O

OH

OH

O

CH2OH

O

OH

OHn

CH2OH

O

# Molecular weight of pullulan : ca. 100,000# 1.0~2.0 cholesteryl groups per 100 glucose units is substituted

References1) K.Akiyoshi and J.Sunamoto, Supramolecular Science, 3, 157-163 (1996)2) H.Shiku, et al, Cancer Chemotherapy and Pharmacology, 46, S77-S82, JUN(2000)

Structure of PUREBRIGHT CP-100T

48

CP-n (Protein) Complex CP agg + n (Protein)

K

Globular Protein

CP-100TFlexible nanoparticle with

hydrogel structure

20~30nm

(1) CHP enhances the incorporation efficiency of cancer-specific antigenic proteins into antigen presenting cells.

(2) Antigen presenting cells that incorporated CHP complexes migrate to lymph nodes spontaneously and present the antigen.

(3) The antigen presenting cells activate T cells. CHP especially increases the number of CD8+ T cells.

(4) The CD8+ T cells attack cancer cells, which have the antigenic protein, and reduce the size of the cancer tissue.

tumor

(1) Presentation to antigen-presenting cells

(2) Migration

(3) CD4+Tcells

(3) CD8+Tcells

(4) Cytotoxicity

Cancer-specific antigen +CHP

CD40LCD40L

Lymph node

Activationclass II

class II

class I

class I

Proposed adjuvant activity of CP-100T particles loaded with antigene

Clinical Studies is using CHP-HER2 (Prof. Shiku , Department of Medicine, Mie University, Japan)

49

Phase I clinical study led by physicians in humans.

CHP-(146Her2) complexes were used in a safety and efficacy study.Safety:Test conditions: 300 µg of CHP-Her2 complexes was parenterally injected at intervals of two weeks.Results: Safety of CHR-Her2 complexes was confirmed.Antibody Titer:

Antibody titers against 146Her2 in blood were measured using the ELISA method, and increasesin antibody titers was confirmed.

Antibody expression:Presence of the antibody against 146Her2 in the blood was confirmed using the Western blottingmethod.

Activation of CD8+ T cells:Number of CD8+ T cells were confirmed to increase as well as CD4+ T cells in vaccinated subjectsby flow cytometry.

Tumor inoculation

4 days 1 week 1 week sacrifice

Count the number of metastases to the lungs

uCMS5m-HE

i.v.

Murine Tumor treatmet using with and withoutCHP-HER2 lmmunized mice

Num

ber

of m

etas

tase

s to

the

lung

s

CHP-HER2treated DC

none DC only HER2p63pulsed DC

200

100

0

CHP-HER2/DCHER2p63/DC

DC onlynone

13

24

*Pre-clinical studies

CHP-(146Her2) complexes were used in a solid tumor mouse model in vivo.CHP-HER2 complexes effectively suppressed metastasis of cancer cells to the lungs.

50

VII. MPC Polymers

(PUREBRIGHT® MB Series)NOF is developing water-soluble polymers (PUREBRIGHT MB) to dissolve drugs of low aqueoussolubility. Polymers comprise 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl meth-acrylate (BMA).

水に難溶性の医薬品を可溶化させることのできるユニークな水溶性ポリマー(PUREBRIGHT MB)を開発しました。2-メタクロイルオキシホスホリルコリン（ＭＰＣ）とn-ブチルメタクリレート（ＢＭＡ）とのコポリマーで構成されていますが、疎水性のＢＭＡユニットを導入することにより疎水結合を介して可溶化されます｡

CH3

(CH2

CH2

CH2 OPOCH2CH2N+(CH3)3

C)

O

C=O

CH3

C=O

O

C4H9

(CH2 C)

O-

O

nm

MPC unit(Hydrophilic Unit)

BMA unit(Hydrophobic Unit)

MPC ; 2-Methacryloyloxyethyl phosphorylcholine BMA ; n-Butyl methacrylate

Structure of NOF’s MPC polymer

How to useSTEP 1 Prepare 30mg/ml of PUREBRIGHT MB aqueous solution(Concentration can be varied ac-

cording to the solubility of drugs). Warm aqueous solution to 60˚C in case not dissolve com-pletely.

STEP 2 Add suitable amounts of drugs into PUREBRIGHT MB solution with stirring.STEP 3 Warm the solution at 60˚C for 5 minutes(Temperature and period of time may be varied

according to the solubility and stability of drugs)

使用方法Step１ 30mg/mlの濃度になるようにPUREBRIGHT MBの水溶液を作成する（濃度は薬剤の溶解性に応じて増減する）。溶けにくい

場合は60℃に加熱し、完全に溶解させる。Step2 Step-1で作成したPUREBRIGHT MBの水溶液を攪拌しながら、希望濃度になるように薬剤を添加する。Step3 60℃で5分間、加熱処理をする（温度と時間は薬剤の熱安定性と溶解性に応じて増減する）。

Product name

PUREBRIGHT MB-37-50T

PUREBRIGHT MB-37-100T

For parenteral use

For oral use

51

We would like to describe toxicological data as follows.

1. Toxicological DataPUREBRIGHT® MB-37 series show only slight hemolization in comparison with other solubilizerssuch as poloxamer and polyoxyethylene castor oil. LD50 are obtained as more than 25,000mg/kg(mice, orally) and more than 1000mg/kg, 8rats, iv).

2. Solubilizing abilityPUREBRIGHT MB-37 series can dissolve poorly water-soluble drugs in aqueous medium.(Refer to Section VII, PUREBRIGHT SL-110)

Toxicological Information of PUREBRIGHT MB-37 Series

Acute toxicity (100T**) Oral LD50 ( Mice ) > 25,000 mg/Kg

Acute toxicity (50T*) Intravenous injection LD50 ( Rats ) > 1,000 mg/Kg

Antigenecity (50T*) Negative

Mutagenecity (50T*) Negative

Skin sensitization (100T**) Non skin sensitization (Guinea pig)

Skin toxicity (100T**) Non-primary irritation (Rabbit)

Eye stimulative (100T**) Practically non-stimulative to eye mucosa (Rabbit)

Hemolysis (50T*) Refer to next figure

Hem

oly

tic

Rat

io (

%) 1 hour 2 hour

4 hour

MB37 Poloxamer 188 Polyoxyl caster oil

Effect of Solubilizing Agent on Hemolytic potential

Red blood cells(guinea pig) are exposed to test compounds (5%) for each hours, and the hemolyticratio is determined by absorbance of 576nm. References:

1) K.Ishihara, et al., J.Biomed.Mater.Res. ,65A, 2. 210-215 (2003)2) T.Konno, et al., Proceed.Int'l. Symp. Control.Rel.Bioact.Mater., 29, 464 (2002)

52

VIII. Hydrophobic Drug Solubilization Kit

(PUREBRIGHT® SL Series)NOF CORPORATION has developed a new Solubilization Kit for poorly water-soluble drugs using ourvarious sophisticated drug delivery technology. Drug solubilizing procedure is simple, facilitatingNCE screening or toxicological testing.

当社はさまざまなDDS技術を結集し、難水溶性薬剤の可溶化キットを開発しました。キットの使用方法は極めて簡単ですので、安全性試験など、新規開発薬剤の初期テストのスクリーニングを容易に行うことができます。

PUREBRIGHT SL-000-S(2 vials of each: total 10 vials)

Content:PUREBRIGHT SL-110: MPC polymer basedPUREBRIGHT SL-220: PEG-phospholipid basedPUREBRIGHT SL-310: PEG-cholesterol based1PUREBRIGHT SL-350: PEG-cholesterol based2PUREBRIGHT SL-411: Phospholipid complex

basedBulk supply also available.

(1) (2) (3) (4) (5) (6)

< Preparation >The following instructions describe preparation of 5 vials of test solution at once.(1) Weigh drugs into test tube or cuvette according to desired concentration in the finished solution.

If you wish to make 1mg/mL finished solution, weigh 12mg of drug, add 3mL of ethanol (selectother solvent when drug is not soluble in ethanol) into test tube, and then dissolve completely bygentle shaking.

希望の最終濃度の4倍濃度の薬剤/溶剤溶液を調製する。例）希望最終濃度　1mg/mLの場合、4mg/mLのエタノール溶液を調製する。

(2) Take 500 µL of drug solution with pipette, and pour the solution into each vial of PUREBRIGHT SLKit respectively.

PUREBRIGHT SLバイアルに(1)で調製した溶液を500μL加える。

(3) Dissolve them completely with shaking. Warm the vial to 50˚C* to achieve a clear solution.

十分に攪拌し、完全に溶解させる。溶解しにくい場合は50℃程度まで加温して溶解させる。

53

Drug Solubilization with PUREBRIGHT® (薬剤の可溶化例)

PUREBRIGHT & Polyoxyl 35 Castor oil : 3% (w/w)

Pep: Peptide type Drug S: SolubleIND: Indomethacin A: Almost solubleMPS: Medroxyprogesterone acetate I : InsolubleCLF: Clofibrate N: No dataNFD: NifedipinePRB: ProbucolCLM: Chlormadinone acetatePTX: Paclitaxel

(4) Evaporate the solvent from the vial according to one of the following methods.a) Place the vial into vacuum chamber previously warmed to 50˚C*, then evaporate the solvent

under vacuum.b) Evaporate the solvent with N2 gas at 50˚C where vials are placed in a warm bath.

Please be careful with handling to avoid the fire or accident with volatile solvent.

エバポレーションにより溶媒を除去する。例１）50℃程度まで加温しながら真空乾燥機でエバポレートする（引火性のある溶媒を用いるときには窒素ガスを流す等して充分に注意してください）。

例2）50℃程度まで加温しながら窒素ガス等によりエバポレートする。

(5) Add 2 mL of deionized water into the vial, and the dissolve completely with shaking.Add 1.8 mL of deionized water in case of using PUREBRIGHT SL-411.

イオン交換水を2mL（PUREBRIGHT SL-411は1.8mL）添加後、十分攪拌を行い、可溶化させる。

Drug in aqueous solution (e.g. 1mg/mL) is now ready.

1mg/mLの濃度の薬物溶液の完成。

Remarks: *Temperature could be changed depending upon drug stability.

注）温度は薬剤の熱安定性により調節してください。

Drug Pep IND MPS CLF NFD PRB CLM PTX

Concentration (mg/mL) 6.0 2.5 0.75 7.5 2.5 5.0 0.5 1.0

PUREBRIGHT SL-110 I I S I I A I S

PUREBRIGHT SL-220 S S N I I I I S

PUREBRIGHT SL-310 S S S S S I S S

PUREBRIGHT SL-350 I I N N I I I S

PUREBRIGHT SL-411 S N N N N S N N

Polyoxyl 35 Castor oil I I I A I I I I

54

IX. High purity CHITOSANs for DDS

(CHITOSAN GH Series)Chitosan is a cationic polysaccharide obtained from arthropod exoskeleton, consisting of linearpoly(1,4) N-acetyl-D-glucosamine corresponding to the deacetylated form of chitin. It is applied indiverse industrial areas such as food, cosmetics and agricultural additives. Other chitosan applica-tions such as hydrogels or gene carrier are published recently. Chitosan has rarely been consideredfor pharmaceutical use due to large amounts of endotoxin contaminants. Using sophisticated purifi-cation technology, NOF and Yaizu Suisankagaku Industry CO., LTD. (YSK) have succeeded in produc-ing chitosan hydrochloride CHITOSAN GH-400EF in which endotoxin levels are remarkably low.

キトサンは、キチンの脱アセチル化体で、グルコサミン残基がβ1-4結合により直鎖状に連なった構造をもつカチオン性の高分子多糖類です。その物理的・生理的機能性を利用して食品原料、化粧品原料、工業原料、農業資材等様々な分野で応用されています。また医薬品分野においても、キトサンの持つユニークな物性が近年注目を集めており、ハイドロゲルや遺伝子導入用キャリヤーなどの各種の応用例が発表されてきています。一方キトサンはエンドトキシンを吸着しやすい性質から、これまで医薬品分野での応用においては支障がありました。NOFと焼津水産化学工業株式会社(YSK)は独自の製法により、エンドトキシン量を大幅に低減化した医薬品グレードのキトサン塩酸塩「CHITOSAN GH-400EF」を開発しました。

O

O

OH

CH2OH

O

O

OH

CH2OH

OO

O

OH

CH2OH

CHITOSAN GH-400EF is obtained by further purification to remove endotoxin from food additivequality chitosan, produced in a sanitized factory starting from shells of pandalus borealis capturedfrom the North Atlantic Ocean near Norway.

CHITOSAN GH-400EFは、ノルウェイ沖北大西洋上で捕獲される北極エビPandalus borealis の殻を出発原料として、衛生管理の行き届いた工場で製造された食品用キトサンを原料として、独自の製法により精製し、エンドトキシンを低減しています。

CHITOSAN GH-400EF

50EU/g

Generic Chitosan (Industrial Grade)

2,300EU/g

Typical Residual Endotoxin

Specifications

ITEM SPECIFICATION TEST

Matter insoluble in water NMT 0.5% EP

pH 4.0~6.0 EP

Viscosity NMT 100 cps NOF

Degree of deacetylation NLT 80% EP

Chlorides 10.0~20.0% EP

Heavy metals NMT 40ppm EP

Loss on drying NMT 10% EP

Sulphated ash NMT 1.0% EP

Endotoxin NMT 100 EU/g NOF

55

Molecular Weight: 612.89 Appearance: White PowderPurity: NLT 99%(HPLC)

CHITOSAN GH-003

CHITOSAN GH-003 is a short oligo-saccharide whose structure contains ß-1,4 bonds linking 3 D-glucosamine molecules shown below. Our high purity (>99% determined by HPLC) CHITOSAN GH-003 is more stable against degradation than ordinary chitosan oligosaccharides due to its open ringstructure, and furthermore, our NOF product is suitable for pharmaceuticals, cosmetics and labora-tory reagent use.

CHITOSAN GH-003は、D-グルコサミン残基3分子がβ-1、4結合したキトサンオリゴ糖です。本製品は糖アルコール体である為、通常のキトサンオリゴ糖よりも物質安定性が向上しており、更に多段階に渡る精製により高純度化（HPLC純度99％以上）しているため、研究用試薬、医薬品、または化粧品原料等の分野でご利用頂けます。

HO

O

2

OH

CH2OHOH

OH

CH2OH

OOH

CH2OH

OOOH

Structure of CHITOSAN GH-003

Reference: Dynamic Contrast-Enhanced MRI of Implanted VX2 tumor in rabbit muscle; Comparisonof Gd-DTPA and NMS60, Magnetic Resonance Imaging, 17, pp. 1297-1305, (1999)

56

X. Biocompatible PEG Anchors for cell membrane <BAM>

(SUNBRIGHT® OE Series)NOF has developed Biocompatible Anchor for cell Membrane (BAM) mimics consisting of oleyl groupsas a hydrophobic cell membrane anchor and activated polyethylene glycol (PEG) for increasing wa-ter solubility and reacting with either physiologically active substances (PAS) or material surfaces atthe PEG terminal. Using BAM, membranes of cells or tissues can be modified with PAS such asproteins, enzymes or drugs without causing damage. Utilizing our new technologies, cells or tissuescan be also immobilized alive on surfaces of various kinds of materials. Possessing excellent func-tions beyond conventional concepts, our new technologies should provide wide application in phar-maceuticals and cosmetics.

細胞にダメージを与えない細胞膜修飾剤（BAM : Biocompatible Anchor for cell Membrane）を開発しました。BAMは細胞膜へのアンカーとして脂質オレイル基と、水溶性を高めるためのポリエチレングリコール（PEG）からなり、生理活性物質や材料表面に結合させるためPEG鎖末端に各種の反応性基を導入した構造を有します。BAMを用いることにより、細胞や組織にダメージを与えることなく、細胞や組織の表面を蛋白質や薬剤などの生理活性物質で修飾することや、細胞や組織を生きたまま各種材料表面に固定化することが可能となりました。従来のコンセプトを超えた機能を持っており、医薬品、化粧品分野で広い用途が期待されます。

Product name

SUNBRIGHT OE-020CS

SUNBRIGHT OE-040CS

SUNBRIGHT OE-080CS

Reactive Group (X)

-CO-CH2CH2-COO-NHS

Mw

2,000

4,000

8,000

Oleyl-O-PEG-X = BAM

PEG chain X:Reactive groupOleyl group

XO

O

n

Physiologically active

substance

(Protein, enzyme, Drug etc)

BAM-Protein

Conjugate

Biocompatible Anchor for cell Membrane (BAM)

57

Application I : Anchoring of BAM-Protein conjugate to the cell membrane

Application II : Anchoring of cell on the BAM modified surface

BAM-Protein Conjugate

BAM-Protein Protein

Cell Cell

CONCEPT micrograph

Anchoring Protein

Cell

Cell membrane

Cell

BSA layer

BAM modified surface

BSA+BAM BSA

CONCEPT micrograph

58

New Non-adherent Cell immobilizing Culture Dishes

BAMCOAT DI-035G

1) New technology for cell immobilizing systems without cell damage.

2) Easy-to-use pre-coated glass bottom dishes.3) Available for numerous applications for observation of non-adherent cells in culture.

ex. Observation of intracellular calciumObservation of Green Fluorescence Protein(GFP) localization

参考文献1) K. Kato, et. al, Biotechnol. Prog., 20, 897-904 (2004)2) K. Kato, et. al, BioTechniques 35,1014-1021 (2003)

59

LIPONIZER LP-90-500(For Small Scale)

Manufacturing System

LIPONIZER LP-293

XI. LIPONIZER® for Liposome production

In order to reliably produce liposomes with certain size distributions, NOF would like to recommendour LIPONIZER. By altering filter sizes, desirable sizes of liposomes (200nm) can be resproduciblyobtained. NOF has currently two sizes of LIPONIZERs. LP-90 for small scale (90mm diameter) andLP-293 for larger scale (293 mm). Specification and pictures are shown below.

Technical Specifications:Max. Pressure 30kgf/cm2

Approx. flow rate 300ml/minMax liquid concentration 200mg/mlTemperature range 5-80˚C

Product Specifications:Weight 230kgMax. Pressure 30kgf/cm2

Approx. Flow Rate 100ml/minMax. Liquid Concentration 300mg/mlTemp. Range 5-80˚C

LIPONIZER® is registered mark of Nomura Micro Science Co., Ltd.

Wah Sin Industrial Pte Ltd519, Balestier Road, #02-02, Singapore 329852Tel: +65 6252 5647 Fax: +65 6256 4883www.wahsin.com