@misc{oai:az.repo.nii.ac.jp:00003337,
 author = {小林, 和子},
 month = {2013-02-28, 2014-08-19},
 note = {The incidence of deep-seated mycosis has been on the increase recently. This type of mycosis often causes serious or fatal infection in patients with a weakened or compromised immune system. For patients who are suffering from deep-seated mycosis, their general health has often deteriorated prior to the development of their fungal infection. Therefore, the drugs used to treat their mycosis need to be effective via intravenous administration and should also have a low toxicity. It is, however, very difficult to produce a drug that possesses a selective toxicity targeted against fungi as both mammalians (the human host) and fungi are both eukaryotes. Compared with anti-bacterial drugs, the development of drugs for the treatment of deep-seated mycosis lags far behind with regard to both efficacy and safety. At the present time, most of the anti-fungal drug treatments are applied orally and treatments are usually performed in the health clinic setting. The relatively safe drugs cover only a narrow anti-fungal spectrum and often induce drug resistance. The antifungal drugs that are considered to be relatively effective in general have the dual disadvantage of having a narrow safety margin and are usually quite difficult to dispense via i.v. infusion due to their lipophilic, water-insoluble characteristics.
Taking the above issues into consideration, a water-soluble azole derivative, 4-[1-[(3-methylaminoacetoxymethyl-pyridin-2-yl)-methyl-carbamoyloxy]-ethyl]-1-[(2R,3R)-3-[4-(4-cyanophenyl)-thiazol-2-yl]-2-(2,5-difluoro-phenyl)-2-hydroxy-butyl]-1H-[1,2,4]triazol-4-iumchloride hydrochloride (hereinafter referred to WSA) has recently been developed. The active form of this compound was not water-soluble, but it was modified with the addition of quaternary ammonium salt to produce a soluble prodrug, which is both injectable and applicable orally. Following administration, this drug is quickly hydrolyzed enzymatically into its active metabolite.
This study examined the safety of the continuous intravenous infusion of this drug as well as its oral administration. Also discussed are the bioavailability of WSA using new toxicological and pharmacokinetic approaches and the introduction of a new method of drug efficacy evaluation.
This examination also includes a toxicological investigation of WSA in monkeys as well as in rodents. To compensate for the possible species difference in the metabolic pathways for this drug (prodrug), and also considering the toxic potential of the liver and secretory system (as is encountered with existing azole analogues), monkeys were employed with the expectation of a more accurate prediction of WSA safety, since the metabolic system of monkeys is very similar to humans. As one of the objectives of this study was to examine the bioavailability and safety of WSA continuous i.v. infusion in humans, it was considered reasonable and pertinent to conduct this study utilizing the monkey. The monkey is the species that is the closest approximation to humans from the viewpoint of both physiology and toxicology.

This study was conducted with the consent of the ethical committee for animal experiments in Nippon Roche K.K.

In the first chapter, the evaluation of repeated oral dose toxicities on the existing azole analogues, fluconazole, itraconazole and ketoconazole was performed.
Although fluconazole possesses a narrow spectrum, it is the most frequently used drug for both oral and intravenous treatment. The study on fluconazole was performed as 2-week and 4-week toxicity studies in SD-Slc rats and a 2-week toxicity study with cynomolgus monkeys.
For itraconazole, a highly lipophilic compound that is frequently utilized for oral treatment, a 2-week toxicity study was undertaken using cynomolgus monkeys.
Using ketoconazole, a drug that has not yet been approved in Japan for human treatment (except for external application) because of its severe toxicity, a 2-week toxicity study was conducted in SD-Slc rats.
The drug dosage utilized in the toxicity studies were as follows:
 Fluconazole: Rat, 2-weekstudy 30, 100, 150, 450 mg/kg/day
 Rat, 4-weekstudy 10, 30 mg/kg/day
 Monkey, 2-weekstudy 15, 100 mg/kg/day
 Itraconazole: Monkey, 2-weekstudy 15, 40, 100 mg/kg/day
 Ketoconazole: Rat, 2-weekstudy 50, 100, 200 mg/kg/day
Statistical analysis (Pitman test: Lehman, E.L., 1974) was applied to the measured data collected in this chapter from hematology values, blood chemistry results and organ weight values only when the treatment group consisted of 3 or more animals (Statistical significance: p ≦ 0.05).

The data obtained in this chapter indicates that these azole analogues adversely affect primarily the liver (increased weight, hypertrophy of hepatocytes, fatty changes) and the adrenal (increased weight, increase of lipid droplets in the cortex, etc.). In addition, the affects of fluconazole treatment were focused mainly on the liver, but itraconazole and ketoconazole tend to affect the adrenal more severely. The comparison between the rats and the monkeys, as the experimental subjects indicated that the toxic characteristics were very similar in both species. The primary differences observed between the two species was the toxicity exhibited in the fluconazole group occurred at much lower drug concentrations in monkeys.

More detailed results that were obtained from chapter one are as follows.
Fluconazole: In the rat study, liver toxicity was detected at 30mg/kg/day and higher for the 2-week administration group and at 10 mg/kg/day and higher for the 4-week administration group. In the high dosage group (30 mg/kg/day) of the 4-week rat treatment group, adrenal toxicity was also uncovered. In the 2-week monkey treatment group, liver toxicity was observed in both dosage groups (15 and 100 mg/kg), and adrenal toxicity was found only in the highest (100 mg/kg/day) group.
Itraconazole: In the 2-week monkey treatment group, toxic changes were detected in the adrenal (40 mg/kg/day and higher) and in the liver for the 100 mg/kg/day group (increased triglyceride: 15 mg/kg and higher).
Ketoconazole: In the 2-week rat study, liver toxicity was observed in the 200 mg/kg/day group, and adrenal toxicity was observed in all dosage groups (50 mg/kg/day and higher).

In the second chapter, the efficacy, bioavailability and safety of WSA, utilizing monkeys, rats and mice as the experimental models was investigated.
In the first section, the results of both single dose and repeated WSA treatment toxicity studies are examined. In the second section of this chapter, the blood concentration levels of the active WSA metabolite are determined and subjected to toxicokinetic analysis. In the third section, a new evaluation method was introduced to facilitate comparisons between WSA and the other effective, but insoluble antifungal drugs, focusing on their efficacy and toxicity when the administration is intravenous.
The first section of the second chapter discussed the results obtained from the toxicity studies on WSA treatment in rats and monkeys. The treatment protocol was as follows:
 Single oral treatment: rat, monkey 500, 1,000, 2,000 mg/kg
 Single i.v. dosing (0.1 mL/body/min.): rat 10, 20, 40 mg/kg
 Single i.v. infusion (6.25 or 8.33 mL/kg/h): monkey 90, 120 mg/kg
 2-week repeated i.v. Infusion: rat, monkey 10, 30, 60 mg/kg/day
 (6.25 or 12.5 mL/kg/h, 2 h/day)
 4-week repeated oral treatment: rat, monkey 10, 30, 90 mg/kg/day
The results obtained from this section are discussed as follows:
Rats: From the data collected, the lethal dosages in the single treatment study were very high, specifically 40 mg/kg (i.v.) and 1,000 mg/kg (oral). In the repeated WSA administration studies, no animal expired from either the oral treatment group (highest dose: 90 mg/kg/day) or in the i.v. infusion group (highest dose: 60 mg/kg). In the 2-week repeated infusion study, the non-toxic level was estimated to be 10 mg/kg/day. An increase in the liver weight values was observed in the 30 and 60 mg/kg/day groups, but accompanying histopathological changes were not detected. In the 4-week repeated oral treatment study, no significant changes were observed in either the liver or adrenal for the 30 mg/kg/day group. Therefore, the non-toxic dosage level was set at 30 mg/kg/day. For the 90 mg/kg/day group, hepatotoxicity (increased liver weight and hypertrophy of hepatocytes) was detected.
Monkeys: The WSA lethal dosages determined for in the single treatment studies were 64 mg/kg (i.v. bolus), 120 mg/kg (i.v. infusion) and 2,000 mg/kg (oral). In the 4-week repeated oral treatment study, expiration of a few of the monkey animal models occurred in the 90 mg/kg/day group, and a slight liver toxicity (increased liver weight and hypertrophy of the hepatocytes) was observed in the 30 mg/kg/day animal group. In the 2-week repeated i.v. infusion study, toxic effects were encountered in the examination of the liver and adrenal from both the 30 and 60 mg/kg/day groups. The non-toxic WSA dosage was estimated to be 10 mg/kg/day for both the oral (4 weeks) and the i.v. (2 weeks) studies.
When compared with fluconazole, the results of these rat studies indicate that WSA has significantly weaker and less toxic effects on both the liver and adrenal. WSA's minimum toxic level was higher than for fluconazole. The adverse effects induced by WSA on the adrenal were present only in the long-term administration groups and within dosage level group where hepatotoxicity was demonstrated. The toxic properties and effects of WSA are very similar to fluconazole.
In the monkeys treated with fluconazole and itraconazole over a 2-week period (oral), an increase in the serum triglyceride values was detected. This parameter indicates that these drugs were having a toxic effect on the liver of these animals and the effect was observed in the 15 mg/kg/day and higher dosage groups. In the 4-week oral WSA toxicity study, a similar effect was uncovered, but only in the 30 mg/kg/day group and higher, which supports the claim of a lower toxicity for the WSA treatment.
In the second section of this chapter, a toxicokinetic analysis was performed utilizing the data collected the studies described in the first section. In these studies, the blood concentrations of WSA and its active metabolite were assayed and recorded. The C_max, AUC and Tii2 values were then calculated. The results obtained demonstrated that WSA was rapidly eliminated for the animal species that were evaluated in this experiment, for both oral and i.v. infusion. The C_max and AUC values for the active metabolite exhibited dose-dependent increases. The indicates that the conversion of the prodrug into the active metabolite was rapid. The active metabolite also remained bioavailable for a longer period of time in the monkeys than in the rats.
 Rat T_1/2: 3.5 h (oral) and 5.1 h (infusion)
 Monkey T_1/2: 12.8 h (oral) and 9.8 h (infusion).
These results suggest that a once-a-day treatment protocol with WSA should be both adequate and effective for use in clinical practice. The oral bioavailability of WSA was very good; measured at 62% in the rat groups and 87% in the monkey groups. The fact that the oral administration can achieve nearly 90% of the blood WSA concentration levels reached by i.v. infusion suggests the feasibility of effective oral treatment in clinical use.
The evaluation of the WSA conversion rate from WSA (prodrug) to the intermediate in. plasma (in vitro) indicated that the T_1/2 of in humans was similar to the results from the monkey group, and much higher than for the rat group (T_1/2: humans 0.75 min., monkeys 0.85 min., rat 0.25 min.). These results support the application of monkey pharmacokinetic data as valid predictor for WSA performance in humans. However, the total conversion rate from WSA to the active body of WSA in plasma (in vitro) showed that the T_1/2 of in humans was similar to that in monkeys and rats (T_1/2: humans 1.6 min., monkeys: 1.75 min., rat: 1.41 min.).
In summary, the studies utilizing the monkey groups demonstrated that a protocol of once-a-day administration of WSA (prodrug) was effective in establishing the concentration of the active metabolite to be high enough for both effective and safe treatment of deep - seated mycosis in humans.
The first and second sections of the second chapter have substantiated the following points:
1. WSA administration adversely affects both the liver and adrenals, as was the case with the existing azole analogues. These negative effects were slight, however, when compared to the existing azole.
2. The i.v. infusion treatment using WSA was very safe, even when the affects were compared with the oral administration. The i.v. administration of the existing anti-fungal drugs had induced severe toxicity, but WSA did not cause sever toxic changes.

In the third section of this chapter, a new method for the evaluation of the efficacy of nearly insoluble compounds, which are difficult to administer intravenously at high enough concentration to both be effective and safe, was introduced. Employing continuous i,v. infusion, sufficient data was collected to evaluate the efficacy of these compounds, despite the low concentration of these solutions. To increase the accuracy of this efficacy evaluation, referrals to the histological findings of renal lesions, damage to the target organs, and the incorporation of histological criteria were included in the evaluation. The results of of this new method of efficacy, evaluation as applied to WSA treatment are also discussed in this section.
To establish this efficacy evaluation method, itraconazole and the ICR mouse model of systemic candidiasis were studied over a 14-day treatment. For the efficacy comparison evaluation, WSA or Amphotericin B were i.v. infused into a F344 fischer rat model representing systemic candidiasis or systemic aspergillosis for a period of 7 days. For both of these experiments, the animals were administered one of the test compounds, continuously via i.v., at a low concentration. The infusion was maintained at a fixed rate using a catheter inserted into the external jugular vein. During the treatment period, the blood concentration of each compound was measured and recorded.
Itraconazole, a nearly insoluble drug, was infused into the mouse models at a dosage level of 24 mg/kg/day over a period of 3 days. The blood concentration of this drug was maintained at ca, 0.3 μg/mL. The average survival time was significantly prolonged (1.85 days for the control group, 7.42 days for the itraconazole group). In the treatment group, a decrease with renal bacteria count was confirmed, and the signs indicative of infection were ameliorated. In the efficacy comparison study, no death were encountered during the drug administration period among the F344 fischer rats treated with 10 mg/kg/day of WSA.
Within the kidney of these animals, no signs of infection were detected the end of the 7-day treatment period. The blood levels of the active drug metabolite were maintained above 0.05 μg/mL throughout this infusion period. The ED_50 value (during dosage level with a 50% survival rate) for WSA was estimated to be 5.5 mg/kg for systemic candidiasis and 3.41 Mg/kg for systemic aspergillosis. The minimal effective concentration was estimated to be ca. 0.05 μg/mL. With amphotericin B, some rats expired during the 7-day administration period (candidiasis group at 6 mg/kg/day and aspergillosis group at 8 mg/kg/day). The dead rats, exhibited characteristics of toxicity attributable to amphotericin B in their kidneys and livers. However, the signs of infection were not found in the kidney of these animals.
In the IC R-mouse model attempting to establish a new efficacy evaluation method, we also introduced a new method to evaluate both the safety and effectiveness of treatment drugs at the same time. Specifically, we set up evaluation criteria of the effective blood concentration of the drug and focused on the histopathological findings that were observed in the target organs. This comparative efficacy study of WSA and amphotericin B using the rats models was then subjected to this new safety evaluation method.
In the third section, it was confirmed that the administration via i.v. infusion was helpful for evaluating the efficacy of nearly insoluble compounds, that also have a short half-life. By administering a low concentration solution of a drug, at a constant rate for 24 hours a day, the blood level of the drug can be maintained within a relatively narrow concentration range. Using this type of infusion, the efficacy of nearly insoluble drugs can be better evaluated.

The conclusions reached in this paper can be summarized as follows:
1. It was confirmed that the existing azole analogues (fluconazole, itraconazole and ketoconazole) adversely and severely affected both the liver and adrenals. The target organs for azole toxicity were verified and index of toxicity was established for these drugs which was later used as a reference for the new azole drug, WSA.
2. Confirmation that WSA, which has been modified to a water-soluble prodrug, possessed a potency with higher safety and sufficient efficacy for the treatment of systemic mycosls.
3. New experimental methods for both safety and efficacy evaluation of anti-fungal drugs were demonstrated. This involved a combination of procedures for infusion administration, toxicokinetic and pharmacokinetic aspects, and histopathological assessment of the target organs for both toxicity and efficacy.
4. Based upon the results obtained from the above described approaches, it was demonstrated that the new WSA was safer and sufficiently more effective compared to be the existing anti-fungal drugs.
5. Clinical application using intravenous continuous infusion would be an effective first intensive treatment, followed by oral administration.

The cumulative opinion that we believe is supported by data from this report was that the development of a new water-soluble azole, WSA, which would be administered orally, via bolus injection and by continuous i.v, infusion possesses sufficient efficacy and is safer than its azole precursors., 近年、深在性真菌症は増加傾向にあり、感染抵抗力の低下した患者においては致命的な重症感染症となっているが、その治療薬開発は抗菌活性および安全性の両面から最近感染症と比べて遅れている。特に、静脈内投与の必要な重症患者が多いにもかかわらず、脂溶性が高いため経口投与が中心でかつ毒性の強いものが多いことから、今日、安全で有効性が高い静脈内投与可能な薬物が必要とされている。
　そこで筆者は、難溶性薬物をプロドラッグ化して水溶性物質とすることを提案し、合成された新規アゾール系抗真菌薬、4-[1-[(3-methylamino acetoxymethyl-pyridin-2-yl)-methyl-carbamoyloxy]-ethyl]-1-[(2R,3R)-3-[4-(4-cyano-phenyl)-thiazol-2-yl]-2-(2,5-difluoro-phenyl)-2-hydroxy-butyl]-1H-[1,2,4]triazol-4-iumchloride hydrochloride(以下、WSAと記載)を用い、安全かつ効果的な使用法としてWSAの静脈内持続投与法について検討した。
　本研究は、毒性学的および薬物動態学的アプローチと新しい薬効判定法の導入を加えた新たな視点からその有用性について究明したものである。
　この研究は、日本ロシュに設置する動物実験倫理委員会の承認を得て実施した。

　第1章では、既存のアゾール系薬物を用いた毒性試験をSD-Slc系ラット(以下ラット)、カニクイザル(Macaca fascicularis、以下サル)を用いて行い、その毒性の強さと特徴を明らかにした。既存薬物のうち、抗菌スペクトルは狭いが毒性は比較的低く、経口および静脈内投与の可能なFluconazole(ラット2週間・4週間試験、サル2週間試験)、難溶性のため経口投与で使用されるItraconazole(サル2週間試験)および毒性が強いKetoconazole(ラット2週間試験)をとりあげた。その結果、Fluconazoleではラット肝臓毒性は2週間投与で30mg/kg、4週間投与では10mg/kg以上で、ラット副腎毒性は4週間投与の30mg/kgで認められた。また、サル2週間投与では肝毒性が15mg/kg以上、副腎毒性が100mg/kgで認められた。また、Itraconazoleのサル2週間投与では副腎毒性が40mg/kg以上で、肝毒性が100mg/kg(トリグリセライドの上昇は15mg/kg以上)で認められた。Ketoconazoleのラット2週間投与では、肝毒性が200mg/kg、副腎毒性が50mg/kg以上で認められた。
　既存のアゾール系薬物の主な毒性作用は共通的であり、肝臓(重量増加、肝細胞肥大、脂肪変性)・副腎(重量増加、皮質脂肪滴増加など)に認められた。また、Fluconazoleでは、肝毒性Itraconazole、Ketoconazoleでは副腎毒性の発現しやすいことを立証した。さらに、ラットとサルにおける毒性は本質的には同じであるが、毒性発現用量に差のあることを確認した。
　第2章では、サルおよびラットを用いてWSAの安全性ならびに有効性の検討から新たな薬物療法の提案を試みた。
　第1節では単回投与(経口投与:ラット・サル共に500、1,000、2,000mg/kg、静脈内ボーラス投与:ラット10、20、40mg/kg、サル4、16、32、64mg/kg、静脈内持続投与:サル90、120mg/kg)、反復投与毒性試験(経口投与:サル10、30、90mg/kg:2週間(活性体)、ラット・サル共に10、30、90mg/kg:4週間、静脈内持続投与:ラット・サル共に10、30、60mg/kg:2週間)を行ってWSAの毒性を明らかにした。
　第2節では、ヒトでの安全性をより正確に評価するため、WSAとその活性体のトキシコキネティックス解析を行った。また、in vitroによる血漿中での活性体への変換率も検討した。
　第3節では、抗真菌薬のより安全で有効な投与法を確立するため、従来の薬効判定基準である生存日数に加え、予め感染時の標的臓器(腎臓、肝臓)を明らかにし、その標的臓器における病理組織学的変化も評価に加えるという薬効・毒性を同時に判定する方法を新に確立し、その方法を用いて静脈内持続投与法について検討した。また、WSAの安全性・有効性については非アゾール系だが効果が最も強いAmphotericin Bと比較検討した。なお、試験法の検討では全身性カンジタ症マウスモデルを用い、難溶性のItraconazole(7.2、14.4、24mg/kg/day)を2週間投与した。また、有効性比較試験には全身性カンジタ症および全身性アスペルギルス症ラットモデルを用い、WSA(カンジタおよびアスペルギルス症:3、10、30mg/kg/day)およびAmphotericin B(カンジタ症:0.3、1.5、6mg/kg/day、アスペルギルス症:2、8mg/kg/day)を7日間、1日24時間の定常静脈内持続投与を行ない薬効・毒性を比較した。
　その結果、第1節:WSAの単回投与における致死量(ラット:経口投与1,000mg/kg、静脈内ボーラス投与40mg/kg、サル:経口投与2,000mg/kg以上、静脈内ボーラス投与64mg/kg、静脈内持続投与120mg/kg)は、大きな用量である(毒性の低い)ことが確認された。ラットの反復投与では、経口(最高用量90mg/kg)、静脈内持続(最高用量60mg/kg)投与のいずれにおいても死亡例は認められず、無毒性量は4週間経口投与では30mg/kg、2週間静脈内持続投与では10mg/kgと推定された。一方、サルの反復投与においては、2週間経口投与(活性体)では90mg/kgで肝毒性(臓器重量増加)が認められ、無毒性量は30mg/kgと推定された。4週間経口投与では90mg/kgで死亡例が発現、30mg/kgでは肝毒性(臓器重量増加および肝細胞肥大)が軽度に認められた。2週間静脈内持続投与では30mg/kg以上で肝毒性が軽度に認められた。このことから経口投与(4週間)、静脈内持続投与(2週間)いずれの試験においても無毒性量は10mg/kgと推定された。
　上記の結果から、ラットでは、副腎、肝臓毒性ともにFluconazole(毒性発現量　肝毒性10mg/kg、副腎毒性30mg/kg:4週間経口投与)と比較してWSAの毒性発現用量(肝毒性90mg/kg:4週間経口投与)は大きく、毒作用が軽度であることが確認された。副腎への毒作用はFluconazoleと同様に肝毒性よりも大きい用量・長期間投与で確認された。サルでもFluconazoleおよびItraconazoleは2週間経口投与試験で15mg/kgからトリグリセライドの上昇が認められ肝への影響が示唆されたが、WSAの2週間経口投与では90mg/kgでのみ、また、4週間経口投与、2週間静脈内持続投与においても30mg/kg以上でのみ毒性が認められ、WSAの低毒性が確認された。
　第2節:第1節の毒性試験の血漿試料を用いWSAおよびその活性体の血中最高濃度(C_max)、血中濃度曲線下面積(AUC)、半減期(t_1/2)を測定し、トキシコキネティック解析を行った。その結果、いずれの動物および投与方法においてもWSAは投与後速やかに消失していることが確認された。また、活性体は最高血中濃度(C_max)、血中濃度曲線下面積(AUC)ともに用量相関性のある増加を示し、WSA投与後のプロドラッグから活性体への変換は、速やかにかつ有効になされていることが確認された。活性体の血中濃度持続時間は、ラット(t_1/2:経口投与3.5時間、静脈内持続投与5.1時間)に比べサルでは比較的長く(t_1/2:経口投与12.8時間、静脈内持続投与9.8時間)、サルのデータから、ヒトへの臨床応用において1日1回の投与でその効果が期待された。また、経口投与でのバイオアベイラビリティーは極めて良好で、ラットでは62%、サルでは87%であり、特にサルでは経口投与によっても静脈内投与の場合の約90%近い血中濃度が得られることが立証され、経口投与による治療も効果的に行えることが確認された。また、in vitroにおける肝細胞での活性本体への変換率は、ラットでは変換時間が短いがヒトとサルは近い値(WSAのt_1/2:ヒト0.75、サル0.85、ラット0.25時間)が示されたことから、薬物動態プロファイルは、ヒトへの外挿性の点ではサルが比較的近いものであることが推察された。
　第3節:マウスを用いた試験では、Itraconazoleの24mg/kg/day投与(血中濃度約0.3μg/mLを維持)で、薬効を示す従来の指標とされる平均生存日数に延長が確認された(平均生存日数:対照群1.85日、24mg/kg/day郡7.42日)。また、病理学的評価により菌の増殖抑制を示唆する変化(観察視野内の菌脚数減少、菌糸出現頻度の減少、菌塊周囲の炎症性細胞浸潤の軽減化など)が肝臓、腎臓で14.4mg/kg/day以上の用量から用量依存性の変化として確認され、この病理学的評価は感度の高い指標であることが立証された。しかし、Itraconazoleの薬効用量24mg/kg/dayは、50%致死量(LD50)[LD50:iv♂46mg/kg、♀40mg/kg(H.V.Cauteren et al., 1991)]の約1/2用量で1日当たりの投与用量としては極めて高用量であるが、血中濃度を有効濃度(約0.3μg/mL)に保つことでこの投与法の採用は毒性を低減させ、かつ効果を強化して薬効評価の行えることを立証した。
　一方、カンジタ症およびアスペルギルス症ラットモデルにおいて、WSAは10mg/kg以上の投与で死亡例は認められず、腎臓、肝臓における真菌病巣は消失し感染症治癒が確認された。また、腎臓、肝臓には薬物に起因したと考えられる変化も認められなかった。Amphotericin Bは、全身性カンジタ症ラットに6mg/kg、全身性アスペルギルス症ラットに8mg/kgの投与(いずれも最高用量)でのみ、肝臓、腎臓に真菌病巣は確認されず薬効も確認されたが、薬効量と毒性量が同一であることが立証された。
　WSAの活性体血中濃度は、薬効の認められた10mg/kg投与では投与期間(7日間)を通し0.05μg/mL以上が維持され最小有効濃度と推察された。3mg/kg投与では、初めの3日間の血中濃度が0.02μg/mLと有効濃度に達しなかったため効果不充分であったと考えられる。
　このように難溶性薬物も含めて安全性と有効性を同時に比較評価するため24時間定常静脈内持続投与法を採用し、被験薬の血中有効濃度および標的臓器の病理学的所見により抗真菌作用および毒性を評価する新しい組み合わせの評価法を確立し、この方法を用いてWSAの有用性比較試験を行い、WSAはAmphotericin Bに比較して安全性が高く有効な抗真菌薬であることを立証した。また、この投与方法は薬物の血中濃度を毒性用量以下に保ち長時間投与できるため毒作用の軽減が可能であること、感染初期の血中濃度が重要であることも確認された。
　第3章では、第1章、第2章の結果をもとにWSAの安全性についてヒトへの外挿性も含めて考察し、ヒトでは1日1回0.6-1mg/kgを投与することで十分な効果が期待できると推察された。また、最小有効血中濃度(0.05μg/mL)、無毒性量(サル静脈内持続投与10mg/kg)におけるC_max(2.93μg/mL)を考慮するとWSAは安全域の広い薬物であることが示された。本薬物は、既存のアゾール系薬物に比較して低い投与量で薬効を発揮し、かつ薬効は低い血中濃度で認められ、さらに毒性はより高い用量でのみ確認されるという極めて安全域の広い薬物であることが提示された。
　本研究の結論は、以下のように要約される。
1. 水溶性プロドラッグとすることで可能となった静脈内持続投与法を用い、薬効が認められ、かつ毒作用の認められていない血中濃度を維持することが可能となった。これにより、毒性を軽減した状態で有効性を維持できることが確認され、安全域を拡大できることを立証した。
2. 安全性と有効性を同時に評価するため、新しい評価試験法を確立した。すなわち、低濃度薬液の静脈内持続投与(24時間持続投与法)、有効血中濃度の確認、評価指標として病理組織学的所見を用いる新しい組み合わせの評価法を確立し、難溶性かつ半減期の短い薬物を開発する際のin vivo評価系としても極めて有用であることを立証した。
3. 静脈内持続投与法と経口投与を組み合わせたより効果的な使用法も可能であることを立証した。

 本研究の成果は、水溶性アゾール系抗真菌薬の有用性のついて、WSAを静脈内持続投与することで、より有効に、また経口投与と同様に安全かつ効果的に本薬物を使用できることを、薬物動態学的アプローチと新しい薬効判定法を加えて毒性学的研究から明らかにしたことにある。},
 title = {医薬安全性からみた水溶性のプロドラッグ新規アゾール系抗真菌薬に関する研究 : 静脈内持続投与法による安全性および有効性},
 year = {}
}