This review summarizes the principle and the features of high-performance frontal analysis (HPFA), a novel chromatographic method to determine the concentrations of unbound drugs under drug-protein binding equilibrium conditions, and its application to the study on the plasma protein binding. HPFA uses a "restricted-access" type HPLC column which retains a small molecule in the drugs in the micropores, while a large molecule in the plasma protein is size-excluded. After direct and continuous injection of a sample, the drugprotein binding equilibrium in the sample solution is regenerated in the column, and the constant concentration zone of the unbound drug appears from the equilibrium zone. The unbound drug is eluted as a trapezoidal peak with a plateau. The concentration in this plateau region is equal to that of the unbound drug in the sample solution, and the concentration of the unbound drug can be determined by subsequent on-line HPLC analysis. HPFA allows a simple analysis following direct injection of a sample, and does not cause undesirable drug adsorption on the membrane nor leakage of the bound drug from the membrane, which are often encountered in the conventional ultrafiltration or dialysis method. HPFA allows the simultaneous determination of the concentrations of total and unbound drugs in a single analysis. HPFA can be easily incorporated into the on-line HPLC system. By coupling HPFA with a chiral HPLC column, the concentration of an unbound racemic drug can be determined enantioselectively. The detection limit can be improved dramatically by coupling HPEA with a preconcentration column. Frontal analysis in capillary electrophoresis format (CE/FA) allows us an ultramicro binding assay. The concentration of the unbound racemic drug can be determined stereoselectively by coupling HPFA with a chiral CE technique.

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¹⁴C-E6123をビーグル犬に経口投与した際の吸収，分布，代謝，排泄および代謝物の同定について検討し，以下の結果を得た．
1．全血，血漿および血球中放射能は，投与後それぞれ2.3，3.0，2.0時間に最高放射能濃度(全血:237.2，血漿;230.0，血球;268.5ng eq./ml)に達し，以後二相性を示して減少した．このときのt_1/2^λ₁およびt_1/2は，それぞれ全血で6.3時間および33.9時間，血漿で4.9時間および38.7時間，血球で3.0時間および18.7時間であった．また，AUCは全血，血漿，血球でそれぞれ3952，3745，4167ng eq.·h/mlであった．
2．血漿中未変化体は，投与後1.3時間で最高濃度(88.7ng/ml)に達し，以後t_1/2が1.7時間で速やかに減少した．このときのAUCは330ng·h/mlであった．
3．ビーグル犬における¹⁴C-E6123のin vitroタンパク結合率は，10～1000ng/mlの範囲でほぼ一定で，57～59%であった．また，ビーグル犬に¹⁴C-E6123を投与した後のin vivoタンパク結合率は，投与後0.75時間で約59%で，投与後8時間では約85%となった．
4．経口投与後の大部分の臓器・組織中放射能濃度が，血漿中放射能濃度と同程度かそれ以上であったことより，組織移行性は高いものと考えられた．放射能の臓器・組織からの消失は，眼の一部の組織を除いて速やかであった．
5．投与後2時間の放射能濃度は，標的臓器である肺中において血漿中の1.8倍高かった．また，このときの血漿および肺中の放射能の存在形態はほぼ同じで，未変化体が最も多く存在した．6．尿および糞中への放射能の排泄率は，投与後48時間までに投与放射能量のそれぞれ約42%および47%と排泄は速やかであった．このときの尿および糞中にはそれぞれ少なくとも7種類の代謝物が認められ，未変化体は少なく，尿中にはM3が，糞中にはM2およびM4が主に認められた．
7．ビーグル犬におけるE6123の主代謝経路は，(1)11位のメチル基の水酸化(MIB)，(2)2位の炭素の水酸化，(3)側鎖の加水分解(M3)であると考えられた。また，2位の炭素が水酸化された後，互変異性体としてM1Aが生成し，さらに還元および酸化をうけてそれぞれM2およびM4に代謝されていると考えられた．

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代表的な経口用セフェム系抗生物質であるcefdinir (CFDN), cefteram (CFTM), ceftibuten (CETB) の蛋白結合率を, 健常若年者 (平均28.3歳) および健常老年者 (平均73.3歳) の血清を用いて, 平衡透析法により同一条件下で比較検討した。老年者における経口用セフェム系抗生物質の蛋白結合率は, 若年者と比較して明らかに低下し, 遊離型薬物濃度の上昇がみられた。パルミチン酸あるいはオレイン酸を添加した健常若年者プール血清を用いた検討において, 各経口用セフェム系抗生物質の蛋白結合率は, 遊離脂肪酸の添加量が増すと共に低下した。老年者における経口用セフェム系抗生物質の蛋白結合率低下の要因として, 血清アルブミン値の低下および遊離脂肪酸の組成比の変化が考えられた。セフェム系抗生物質の効果・副作用の発現には遊離型薬物濃度が関与するので, 老年者への経口用セフェム系抗生物質の投与に際しては, このような蛋白結合率の低下に起因する薬理効果の増強, 副作用発現率の増加の可能性にも十分考慮すべきであろう。

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幼若ラットに200mg/kgの¹⁴C標識ロキタマイシン (¹⁴C-RKM) を経口投与し, RKMの幼若動物での体内動態を研究した。
RKMは投与後30分に最高血液中濃度を示し, 平均最高濃度は20.25μg/mlであった。又, その時の血液中濃度曲線下面積 (AUC) は93.23μg・hr/mlであつた。
RKMのin vivoでのタンパク結合率は幼若ラット, 成熟ラット共に約30%であつた。RKMは広く組織に分布し, 肝臓, 腎臓, 肺臓, 脾臓, 膵臓, 骨髄及び顎下腺などに高濃度移行した。
幼若ラットの血漿, 尿及び胆汁中に検出された主な代謝物は10-OH-RKM, Leucomycin A₇, Leucomycin V及び14-OH-Leucomycin Vであった。
RKM経口投与後144時間までに投与量の約97%が尿中及び糞中に排泄された。又, 胆管カニューレを施したラットに十二指腸内投与した場合, 投与後24時間までに投与量の7.42%が尿中へ, 25.66%が胆汁中へ排泄された。
ロキタマイシン (RKM) の吸収, 分布, 代謝及び排泄はすでに成熟ラット及び成犬において検討されている1～3)。
今回, 著者らはRKMの小児科領域での臨床使用に先立ち¹⁴C標識RKM (¹⁴C-RKM) 投与後の幼若ラットにおける吸収, 分布, 代謝及び排泄を検討した。

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In recent years, the clinical development of oligonucleotide therapeutics, such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA)，has been active. Applications for regulatory approval require a series of assessments of the absorption, distribution, metabolism, excretion, and drug-drug interaction characteristics of these oligonucleotide therapeutics using appropriate methods. It is particularly important to understand the tissue distribution and plasma/serum protein binding properties of oligonucleotide therapeutics in assessing their efficacy and safety. However, no comprehensive studies have been conducted to investigate how tissue distribution and protein binding are evaluated and what properties are determined as a result.

In this study, we examined the review reports for approved oligonucleotide therapeutics released by the regulatory authorities, as well as related papers to investigate the evaluation methods and the tissue distribution and plasma/serum protein binding properties of the currently approved ASO and siRNA therapeutics.

First, quantitative whole-body autoradiography (QWBA) studies using radiolabeled compounds were in principle conducted for the evaluation of tissue distribution throughout the whole body, as is the case with small-molecule drugs. In many cases, distribution to tissues of particular interest, such as organs with a high distribution rate, was evaluated by a combination of methods, including liquid chromatography-mass spectrometry (LC-MS)，capillary electrophoresis (CE)， high-performance liquid chromatography (HPLC)，hybridization enzyme-linked immunosorbent assay (ELISA)，and hybridization electrochemiluminescence (ECL) after administration of unlabeled compounds. The results of these tissue distribution evaluations showed that systemically administered ASO therapeutics consisting solely of oligonucleotides were rapidly distributed throughout the body and were highly concentrated in the kidneys in all cases, regardless of animal species. In contrast, all the siRNA therapeutics were highly directed to the liver, and GalNAc-siRNA, in particular, tended to accumulate predominantly in the liver, the therapeutic target tissue.

The plasma/serum protein binding of ASO therapeutics was evaluated by methods commonly used for small-molecule drugs such as ultrafiltration and ultracentrifugation, while gel-shift assay was also used for siRNA therapeutics as a new evaluation method. As regards the protein binding properties of the ASO therapeutics, the plasma/serum protein binding rate of morpholino ASOs was generally low (40％ or less)，whereas the plasma protein binding rate of phosphorothioate ASOs was 85％ or more. In contrast, for siRNA therapeutics, LNP-siRNA showed a low serum protein binding rate of approximately 2％ or less, whereas the plasma protein binding rate in GalNAc-siRNAs at concentrations around the clinical exposure level was 76％ or higher in human.

The tissue distribution and protein binding of oligonucleotide therapeutics are particularly sensitive to the molecular structure of oligonucleotides and the drug delivery system (DDS) technology employed, so an accurate understanding of these properties is important for the development of oligonucleotide therapeutics. This survey revealed that new evaluation methods for tissue distribution and protein binding were employed in addition to conventional evaluation methods, and indicated that these methods provided an improved understanding of the tissue distribution and protein binding properties.

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　近年，創薬研究における開発候補化合物のヒト動態特性の予測は，効率的な医薬品開発に欠くことのできないタスクの一つと考えられている．創薬初期におけるヒト動態予測は，リード化合物の創製，開発候補化合物の選出に有用な情報であり，さらに，前臨床から臨床に移行する局面では，第Ⅰ相試験における開始投与量の妥当性および上限用量の見積りに有用な情報を与える．また，薬効発現および毒性発現における動物種差を考慮する必要はあるものの，開発候補化合物のヒト動態予測と実験動物におけるPK-PDまたはTK-TD解析を統合することにより，前臨床段階で臨床有効用量の見積り，ヒトでの安全域の考察に利用することも可能であると考えられる．一方，前臨床段階でのヒト動態特性の予測は，これまで主流であった実験動物データを用いた方法（アニマルスケールアップ/アロメトリックスケーリング）から臓器・組織を血流で繋ぎ，各部位における薬物濃度変化を数学的に表現する生理学的薬物速度論モデル（PBPKモデル）を用いた方法に移行しつつある．また，PBPKモデルによる動態予測は，生理学的パラメータ（臓器重量，血流量など），化合物の物性情報（分子量，LogPなど）およびin vitro試験結果（代謝安定性，率など）を用いて実施するため，その用途は健常人における体内動態の予測に加え，生理学的パラメータ，薬物代謝能などが著しく変動する病態時（肝機能低下，腎機能低下）および多剤併用時（薬物相互作用の予測）の動態予測まで広がる．
　本発表では，PBPKモデルを用いたヒト体内動態予測の特徴およびその予測精度に関して報告する．また，ヒト体内動態予測の安全性担保を含めた種々局面での使用例を紹介する．

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The transfer of sulfonamides to eggs and its relationship to sulfonamide levels inthe plasma and other tissues were examined in hens fed a diet containing sulfadi-methoxine (SDM), sulfamonomethoxine (SM[NI), or sulfamerazine (SMR).When hens were fed diets containing 0.005% sulfonamide, it was hardly possibleto find both SDM and SMM in both egg-albumen and yolk.When O.2% sulfonamide diets were fed, a rise in sulfonamide level was observed inplasma>egg-albumen>egg-yolk, as listed in the decreasing order of rapidity, with apeak level appearing 2 to 4, 6 to 8, and 8 to 10 days, respectively, after the start of theexperimental feeding. SDM levels in the egg-albumen were nearly a half of those inthe plasma, while SMM levels, as well as SMR levels, were almost the same both in theegg-albumen and in the plasma. In every case of the plasma, egg-albumen and yolk, SDM levels were higher than SMM and SMR levels. The withdrawal of SDM andSMM after a 30-day period of medication accompanied the disappearance of these drugsfrom the plasma, egg-albumen, and yolk after 4, 6, and 10 days, respectively. On theother hand, SMR could not be detected after 4 days in these tissues. The drug levelswere relatively high in the gall-bladder, plasma, and kidney, but were low in the ovary, ileum, and thoracic muscle. The level in the egg-albumen was as high as that in thekidney, while the egg-yolk showed the lowest level of 15 tissues tested. A considerablylarge difference was found in the proportion of acetylated sulfonamide arnong the drugs, as well as the tissues tested.Evidences have been obtained that the differences observed in plasma level andtransferability to eggs among the sulfonamides studied depended more on the durabilityof these drugs and the degree of plasma-protein binding than the rapidity of absorptionfrom the alimentary tract.

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