JP2008503570A - Hiv患者における、障壁の完全性の改善 - Google Patents
Hiv患者における、障壁の完全性の改善 Download PDFInfo
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- JP2008503570A JP2008503570A JP2007517984A JP2007517984A JP2008503570A JP 2008503570 A JP2008503570 A JP 2008503570A JP 2007517984 A JP2007517984 A JP 2007517984A JP 2007517984 A JP2007517984 A JP 2007517984A JP 2008503570 A JP2008503570 A JP 2008503570A
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Abstract
【選択図】なし
Description
EP 745001号は、潰瘍性結腸炎の治療のための不消化性オリゴ糖ならびにn-3およびn-6脂肪酸の組み合わせについて記載している。
a. EPA、DHAおよびARAを、20および22炭素原子を有する長鎖多価不飽和脂肪酸の含量が、全脂肪含量の85重量%を上回らないように含み;且つ
b. 単糖単位で90%未満の相同性を有する、少なくとも二つの異なるオリゴ糖を含む組成物に関する。
a. エイコサペンタエン酸(EPA)、ドコサヘキサエン酸(DHA)およびアラキドン酸(ARA);ここでの20および22炭素原子を有する長鎖多価不飽和脂肪酸の含量は、全脂肪含量の85重量%を上回らない;および
b. 少なくとも二つの異なるオリゴ糖(OL1およびOL2);該二つのことなるオリゴ糖は、単糖単位で90%未満の相同性を有する。
本発明者らは驚くべきことに、エイコサペンタエン酸(EPA、n-3)、ドコサヘキサエン酸(DHA、n-3)およびアラキドン酸(ARA、n-6)が、腸のタイトジャンクションの透過性を効果的に低下させることを発見した。GLA (n-6)もまた、効率的に障壁の透過性を低下させる。それゆえ、本組成物は、腸の障壁の完全性の改善に特に適し、任意にGLAと併用され、EPA、DHAおよびARAを含む。
本栄養性の組成物はまた、十分な栄養を提供するために、好ましくはオメガ-9(n-9)脂肪酸(好ましくはオレイン酸、18:1)を提供する。好ましくは、本組成物は、全脂肪酸の重量を基準にして、少なくとも1重量%のn-9脂肪酸を提供し、より好ましくは少なくとも5重量%で提供する。n-9脂肪酸の含量は、好ましくは80重量%未満である。
本発明による適したオリゴ糖は、少なくとも2単糖単位の重合度を有し、ヒトの上部消化管(小腸および胃)に存在する酸または消化酵素の作用によって、腸にて消化されないまたは部分的にしか消化されないが、ヒトの腸内フローラによって発酵され得る糖である。単糖単位という用語は、閉環構造を有する単位を意味し、好ましくは、ピラノースまたはフラノースの形態といった六炭糖を意味する。オリゴ糖の重合度は、典型的に、60単糖単位未満であり、好ましくは40単糖単位未満であり、さらに好ましくは20単糖単位未満である。
a. (DPが2から5のオリゴ糖):(DPが6、7、8および/または9のオリゴ糖)>1;および
b. (DPが10から60であるオリゴ糖):(DPが6、7、8および/または9のオリゴ糖)>1
は、ともに1より大きい。
障壁の完全性をさらに改善するために、本組成物は、好ましくは2から60のDPを有する酸性オリゴ糖を含む。酸性オリゴ糖という用語は、N-アセチルノイラミン酸、N-グリコロイルノイラミン酸、遊離またはエステル型カルボン酸、硫酸基およびリン酸基から成る群から選択される少なくとも一つの酸性基を含むオリゴ糖を意味する。酸性オリゴ糖は、好ましくはウロン酸単位(すなわち、ウロン酸ポリマー)、より好ましくはガラクトウロン酸単位を含む。酸性オリゴ糖は、均一のまたは不均一の炭水化物であってよい。適した例は、ペクチンおよび/またはアルギン酸塩の加水分解産物である。消化管において、ウロン酸ポリマーは、加水分解されウロン酸モノマーとなり、それが腸内の酢酸塩の生成を刺激し、次に腸内の粘膜分泌を刺激する(Barcelo et al., Gut 2000; 46:218-224)。
Rは、好ましくは、水素、水酸基または酸性基から成る群から選択され、好ましくは水酸基であり;および
R2、R3、R4およびR5から成る群から選択される少なくとも一つは、N-アセチルノイラミン酸、N-グリコロイルノイラミン酸、遊離またはエステル型カルボン酸、硫酸基およびリン酸基、を表し、R2、R3、R4およびR5の残りは水酸基および/または水素を表す。好ましくは、R2、R3、R4およびR5から成る群から選択される一つは、N-アセチルノイラミン酸、N-グリコロイルノイラミン酸、遊離またはエステル型カルボン酸、硫酸基およびリン酸基、を表し、残りは水酸基および/または水素を表す。さらに好ましくは、R2、R3、R4およびR5から成る群から選択される一つは、遊離またはエステル型カルボン酸を表し、R2、R3、R4およびR5の残りは水酸基および/または水素を表し;および
nは整数であり、六炭糖単位(重合度もまた参照、下記)の数を表し、この六炭糖単位は何れの六炭糖単位であってよい。適したnは、1から5000の整数である。好ましくは、六炭糖単位はウロン酸単位である。
すぐ摂取できる液体の形態の場合、本組成物は、不消化性オリゴ糖を、好ましくは1リットル当たり0.1から100グラム含み、より好ましくは1リットル当たり0.5から50グラム含み、さらに好ましくは1リットル当たり1から25グラム含む。高すぎるオリゴ糖の含有量は、過度の発酵のために不快感を引き起こすかもしれず、一方で非常に低い含量は、不十分な粘膜層をもたらすかもしれない。
本組成物は、哺乳類、特にヒトにおける、障壁の完全性の改善のための方法に好都合に使用することができる。本組成物はまた、障壁の完全性の低下に関連する疾病の治療または予防のための方法において好都合に使用することができ、当該方法は、哺乳類に本組成物を投与することを含む。本組成物は好ましくは経口にて投与される。
本組成物は、好都合に、乳児用食および臨床用食といった、食事に応用することができる。そのような食事は、好ましくは、脂質、タンパク質および炭水化物を含み、および好ましくは液体の形態にて投与される。本発明にて使用される「液体食」という用語は、その乾燥食の混合物と適した液体(例えば水)を混合することを示す説明書が添付される、乾燥食(例えば、粉)を含む。
腸管上皮細胞株T84 (American Type Culture Collection (ATTC)、米国、マナッサス)の単層(MC)を、トランスウェルフィルター(transwell filters)(Corning, Costar BV,オランダ)にて培養し、粘膜性および漿膜性の両サンプリングおよびヒト腸管上皮細胞の刺激を行えるようにした。集密状態となった後二週間、単層を、内腔の画分にて、多価不飽和脂肪酸ARA(アラキドン酸;5,8,11,14-エイコサテトラエン酸)、DHA(cis-4,7,10,13,16,19ドコサヘキサエン酸)、EPA(エイコサペンタエン酸)またはコントロールであるパルミチン(C 16:0)酸(Palm)(Sigma、米国、セントルイス)とともに、培養した。後半の手順は、食事性化合物のin vivoの投与経路を模倣するために選択された。細胞を、ARA、DHA、EPA、GLAまたはパルミチン酸で、0、24、48および72時間、異なる濃度で(10μMおよび100μM)培養した。実験は、基底障壁の完全性(basal barrier integrity)を評価するために行った。上皮性障壁の機能は、経上皮抵抗性(TER、Ω.cm2) および4kD FITCデキストラン(paracellular permeability marker, Sigma, 米国)の透過性を測定することによって決定した。該経上皮抵抗性は、上皮のボルト−オームメーター(EVOM; World Precision Instruments, ドイツ)にて測定した。4kD FITCデキストランの上皮の透過性は以下のように決定した。デキストランのフラックスに先駆けて、培地を、1時間かけてフェノールの入っていない培養培地と交換し、続いて5μl(ストック 100 mg/ml)の4 kDa FITC-デキストランを内腔画分に添加した。30分の培養の後、100μlのサンプルを漿膜性画分から回収し、蛍光シグナルを、励起波長を485 nmおよび放出波長を520 nmで測定した(FLUOstar Galaxy(登録商標), BMG Labtechnologies, 米国)。FITC-デキストランのフラックスは、pmol FITC-デキストラン/cm2/hで算出した。統計分析は、ANOVA (SPSS version 10)を用いて行った。
腸管上皮細胞株T84 (ATTC、米国)の単層(MC)を、トランスウェルフィルター(transwell filters)(Corning, Costar BV,オランダ)にて培養し、粘膜性および漿膜性の両サンプリングおよびヒト腸管上皮細胞の刺激を行えるようにした。集密状態となった後二週間、単層を、IL-4(2 ng/ml、漿膜性画分、Sigma、米国)の存在下、多価不飽和脂肪酸ARA、DHA、GLA、EPAまたはコントロールであるパルミチン酸(10μMまたは100μM、粘膜性画分、Sigma、米国、セントルイス)を添加して、または添加せずに培養した。細胞を、IL-4培養に先駆けて、48時間GLA、ARA、DHA、EPAまたはパルミチン酸存在下にて前培養した。IL-4と、PUFA’sおよびパルミチン酸の共培養を、さらに48時間続け;一方で培養培地および添加物を24時間毎に交換した。上皮の障壁の機能は、実施例1に記載した通り経上皮の抵抗性(TER)および透過性を測定することで決定した。統計的評価は実施例1に記載の通りに行った。
微生物を、ミルク(人工栄養)で育った乳児の新鮮な便から得た。生まれて1から4ヶ月の間の乳児から採った新鮮な便の材料を、溜め、保存培地に2時間入れた。基質として、プレバイオティクス(TOS;TOS/イヌリン(HP)混合物9/1 (w/w)の比率;イヌリン;オリゴフルクトース(OS)/イヌリン混合物1/1(w/w)の比率)を用い、または何も用いなかった(ブランク)。トランスガラクトオリゴ糖(TOS)は、Vivinal GOS, Borculo Domo Ingredients(オランダ、ズヴォーレ)から得て、不消化性オリゴ糖として:33重量%の二糖、39重量%の三糖、18重量%の四糖、7重量%の五糖および3重量%の六-、七-、八糖を含む。イヌリン(HP)は、Orafti active food ingredients(ベルギー、ティーエン)より、Raftiline HP(登録商標)として得て、平均DPは23である。培地:McBain&MacFarlane培地:緩衝ペプトン水3.0g/l、イースト・エクストラクト2.5 g/l、ムチン(刷子縁(brush borders)) 0.8 g/l、トリプトン3.0g/l、L-システイン-HCl 0.4 g/l、胆汁酸塩0.05 g/l、K2HPO4.3H2O 2.6 g/l、NaHCO3 0.2 g/l、NaCl 4.5 g/l、MgSO4.7H2O 0.5 g/l、CaCl2 0.228 g/l、FeSO4.7H2O 0.005 g/l。500 ml Scottビンを培地で満たし、121℃で15分間滅菌する。緩衝培地:K2HPO4.3H2) 2.6 g/l、NaHCO3 0.2 g/l、NaCl 4.5 g/l、MgSO4.7H2O 0.5 g/l、CaCl2 0.228 g/l、FeSO4.7H2O 0.005 g/l。K2HPO4またはNaHCO3で、pH 6.3±0.1に調整。500 ml Scottビンを培地で満たし、121℃で15分間滅菌する。保存培地:緩衝ペプトン20.0 g/l、L-システイン-HCl 0.5 g/l、チオグリコール酸ナトリウム0.5 g/l、レザズリン錠剤を1リッター当り1錠。1M NaOHまたはHClで、pH 6.7±0.1に調整。電子レンジで沸騰させる。血清ビンを25 mlの培地で満たし、121℃で15分間滅菌した。
2) 85mg イヌリン
3) 85mg TOS/イヌリン 9/1 (w/w)の比率、および
4) 85mg OS/イヌリン 1/1 (w/w)の比率。
単層の腸の上皮T84細胞(ATCC、米国)を24または96ウェル組織培養プレート(Corning B.V.)にて培養した。T84を、0.025-4.0 mMの濃度範囲にて短鎖脂肪酸酢酸塩、プロピオン酸塩および酪酸塩とともに、24時間培養した。上清および/または細胞を回収し、MUC-2(ムチン)発現を検出した。ムチンは非常に大きな糖タンパク質(500 kDaより大きい)であり、ウエスタンブロッティング技術で扱うには難しいため、ドットブロット技術にて、細胞培養のMUC-2発現を検出した。この方法は、免疫前血清(T84をネガティブに染色)、CCD-18Co (ATCC, 米国)ネガティブコントロール細胞およびウシ血清アルブミン(BSA)を用いて確認した。細胞サンプルをLaemmli(タンパク質単離バッファー)にて回収し、マイクロプロテインアッセイ(Biorad, 米国)を用いて、説明書に従って、タンパク質検出を行った。サンプル(0.3-0.7-1.0μg/2μl)をニトロセルロース膜(Schleicher & Schuell,ドイツ)にドットした。膜を、TBST/5% Protivar(Nutricia,オランダ)でブロックし、続いて抗MUC-2抗体(オランダ、ロッテルダム、エラスムス大学Einerhand博士のご好意による提供)で1時間インキュベートした。洗浄の後、ブロットをヤギ抗ウサギ-HRP(Santacruz Biotechnology,米国)でインキュベートし、基質による検出のために、ECL(Roche Diagnostics,オランダ)を用いた。濃度測定を、Lumi-Imager (Boehringer Mannheim B.V.,オランダ)を用いて行い、シグナルをライトユニット(light units)で表した(BLU)。BLU’sはまた、コントロールのインキュベートに対して相対値で表した(%BLU)。MUC-2発現におけるSCFAの刺激の効果を比較するため、基本のMUC-2発現の値を差し引いた。
成分(リッター当り)、エネルギー672 Kcal;タンパク質15 g;乳清: カゼイン比 60:40; 脂肪 36 g; 炭水化物72 g; ビタミン A 750 RE;混合型天然カロチド(carotids) 400 IU;ビタミン D 10.6 mcg; ビタミン F 7.4 mg; ビタミン K 67.0 mcg; ビタミン B.sub.1 (チアミン) 1000 mcg; ビタミン B.sub.2 (リボフラビン) 1500 mcg; ビタミン B.sub.6 (ピリドキシン) 600 mcg; ビタミン B.sub.12 (シアノコバラミン(cyanacobalmine)) 2.0 mcg; ナイアシン 9.0 mcg; 葉酸80 mcg; パントテン酸 3000 mcg; ビオチン90 mcg; ビタミン C (アスコルビン酸) 90 mg; コリン100 mg; イノシトール33 mg; カルシウム460 Mg; 亜リン酸333 Mg; マグネシウム64 Mg; 鉄8.0 Mg ; 亜鉛6.0 Mg; マンガン50 mcg; 銅560 mcg; ヨウ素100 mcg; ナトリウム160 mg; カリウム650 mg; 塩素イオン433 mgおよびセレン14 mcg;ここにおいて脂肪含量は、魚の油3グラムおよび40%アラキドン酸3グラム(DSM Food Specialties, オランダ、デルフト);さらに、4グラムのトランスガラクトオリゴ糖Elix’or(商標)(Borculo Domo Ingredients, オランダ)および4グラムのRaftiline(商標)(Orafti Active Food Ingredients, ベルギー)を含む。
原材料 g/日 タンパク質 炭水化物 脂肪 g/100g
乳タンパク質 20.00 15.00 2.10 0.80 21.04
卵タンパク質 21.09 16.87 0.00 0.00 22.19
ルリジサ油 4.00 0.00 0.00 4.00 4.21
EPA-DHA 油 6.00 0.00 0.00 6.00 6.31
ガラクトオリゴ糖 15.38 0.00 4.78 0.00 16.18
イヌリン 0.79 0.00 0.00 0.00 0.83
ペクチンハイドロール 8.54 0.11 0.09 0.00 8.98
フルクトース群 15.40 0.00 11.92 0.00 16.20
グリセリン 3.85 0.00 3.83 0.00 4.05
計 95.05 31.98 22.72 10.80 100.00
一日当り 100g当り
kcal En% kcal
エネルギータンパク質 128 40.5 135
エネルギー炭水化物 91 28.8 96
エネルギー脂肪 97 30.8 102
Claims (12)
- ヒト免疫不全症ウイルス(HIV)に感染した患者の治療方法において使用する組成物を製造するための、多価不飽和脂肪酸の使用であって、前記治療方法は、ヒト免疫不全症ウイルスに感染した当該患者にたいして、下記aおよびbを含有する組成物を投与することを含んでなる使用:
a. エイコサペンタエン酸(EPA)、ドコサヘキサエン酸(DHA)およびアラキドン酸(ARA);ここでの20および22炭素原子を有する長鎖多価不飽和脂肪酸の含量は、全脂肪含量の85重量%を上回らない;および
b. 少なくとも二つの異なるオリゴ糖(OL1およびOL2);該二つのことなるオリゴ糖は、単糖単位で90%未満の相同性を有する。 - 前記組成物が、さらにガンマ-リノレン酸(GLA)を含む、請求項1に記載の使用。
- 栄養性の組成物であって:
a. EPA、DHAおよびARAを、20および22炭素原子を有する長鎖多価不飽和脂肪酸の含量が、全脂肪含量の85重量%を上回らないように含み;且つ
b. 単糖単位で90%未満の相同性を有する、少なくとも二つの異なるオリゴ糖(OL1およびOL2)を含む組成物。 - ガラクトオリゴ糖ならびに、フルクトオリゴ糖、イヌリンおよびその混合物から成る群から選択されるフルクタン(fructan)を含む、請求項3に記載の組成物。
- 請求項3または請求項4に記載の組成物であって、少なくとも10重量%のオリゴ糖が、2から5の重合度(DP)を有し、少なくとも5重量%が10から60の間のDPを有する組成物。
- 請求項3から請求項5の何れか1項に記載の組成物であって、さらに、酸性オリゴ糖、好ましくはDPが2から60の間のウロン酸ポリマーを含む組成物。
- 請求項3から請求項6の何れか1項に記載の組成物であって、7.5から12.5エネルギー%のタンパク質;40から55エネルギー%の炭水化物;および35から50エネルギー%の脂肪を含み、前記タンパク質が、加水分解された乳タンパク質、植物性タンパク質および/またはアミノ酸から成る群から選択されたメンバーを含む組成物。
- 請求項3から請求項7の何れか1項に記載の組成物であって、該組成物が、0.6から0.8 kcal/mlのカロリー含量;50から500 mOsm/kgの重量モル浸透圧濃度;および50 mPas未満の粘性を有する組成物。
- 前記組成物がさらにGLAを含む、請求項3から請求項8の何れか1項に記載の組成物。
- 医薬品としての使用するための、請求項3から請求項9の何れか1項に記載の組成物。
- 請求項1に記載の使用であって、前記患者がCD4+ T-リンパ球細胞数が200から700細胞/μl液体であるヒトの患者であり、および前記患者が高活性抗レトロウイルス療法(Highly Active Antiretroviral Therapy)による治療を受けていない使用。
- 下痢の治療または予防の方法において使用する薬物を製造するための、請求項3から請求項9の何れか1項に記載の組成物の使用であって、該方法が請求項3から請求項9の何れか1項に記載の組成物を哺乳類に投与することを含む使用。
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AU2005253898A1 (en) | 2005-12-29 |
BRPI0512528B8 (pt) | 2022-11-22 |
BRPI0512528A (pt) | 2008-03-25 |
PL1758469T3 (pl) | 2010-05-31 |
US20100167982A1 (en) | 2010-07-01 |
EP1758469B2 (en) | 2013-09-25 |
DK1758469T4 (da) | 2013-11-04 |
WO2005122791A3 (en) | 2006-04-13 |
EP1758469A2 (en) | 2007-03-07 |
CA2570208A1 (en) | 2005-12-29 |
JP5301155B2 (ja) | 2013-09-25 |
ES2336015T5 (es) | 2014-01-09 |
DK1758469T3 (da) | 2009-12-21 |
NZ552118A (en) | 2010-03-26 |
BRPI0512528B1 (pt) | 2018-02-06 |
RU2429718C2 (ru) | 2011-09-27 |
ES2336015T3 (es) | 2010-04-07 |
AU2005253898B2 (en) | 2010-02-18 |
DE602005017389D1 (de) | 2009-12-10 |
PL1758469T5 (pl) | 2014-01-31 |
ATE446686T1 (de) | 2009-11-15 |
CN1972604B (zh) | 2010-06-02 |
RU2007102053A (ru) | 2008-07-27 |
NO332430B1 (no) | 2012-09-17 |
SI1758469T1 (sl) | 2010-02-26 |
CN1972604A (zh) | 2007-05-30 |
WO2005122791A2 (en) | 2005-12-29 |
MXPA06015071A (es) | 2007-08-06 |
EP1758469B1 (en) | 2009-10-28 |
US20080015166A1 (en) | 2008-01-17 |
PT1758469E (pt) | 2010-02-03 |
NO20065915L (no) | 2007-03-22 |
SI1758469T2 (sl) | 2013-12-31 |
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