In addition to the well-known role of mitochondria in energy metabolism, regulation of cell death has recently emerged as a major function of these organelles. This, in turn, seems to be linked to their role as the major intracellular source of reactive oxygen species (ROS), which are generated at Complex I and III of the respiratory chain. Excessive ROS production has now been implicated in mtDNA mutations, ageing, and cell death. Although mitochondrial dysfunction can result in ATP depletion and necrosis, these organelles are also involved in the execution of apoptotic cell death by mechanisms which have been conserved through evolution. Hence, many lethal agents target the mitochondria and cause the release of cyto-chrome c and other proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increase in “free” cytochrome c in the mitochondrial intermembrane space. Subsequent release of cytochrome c into the cytosol occurs by pore formation in the outer mitochondrial membrane mediated by pro-apoptotic Bcl-2 family proteins, such as tBid, Bax and Bak, or by Ca2+-and ROS-triggered mitochondrial permeability transition, although the latter pathway might be more closely associated with necrotic cell death. Taken together, these findings have placed the mitochondria in the focus of cell death research.
In an almost every moment, living organisms are subjected to diverse types of stress both external and internal sources. While excessive stress leads to necrotic or apoptotic death, moderate amounts of noxious stimuli may render the cells adaptive to subsequent stress. Such adaptive response to stress normally accompanies de novo synthesis of proteins via activation of distinct stress-responsive signaling. One of the key signaling molecules involved in cellular adaptation or tolerance to a wide array of stressful conditions is NF-κB. Nrf2 is another important redox-sensitive transcription factor that is involved induction of phase-2 detoxifying or antioxidant genes in response to oxidative and electrophilic insults. Many dietary phytonutrients can induce ARE-driven antioxidant/phase-2 detoxifying gene expression, thereby fortifying cellular defence against oxidative insult. Cysteine thiols present in various transcription factors and their regulators function as redox sensors in fine-tuning of transcriptional regulation of many genes essential for maintaining cellular homeostasis. Thus, oxidation or covalent modification of thiol groups present in the above redox-sensitive transcription factors and their regulating molecules can provide a unique strategy for molecular target-based chemoprevention and cytoprotection.
In 1995, ICH S6, which dealt with assessing the safety of biologic drug products, was published. At that time, there was little practical experience with the subject and most of the recommendations were fairly uninformative. The guidance has served the drug development process well, however, primarily because it did point out that some types of studies would not be useful, thus limiting the demand by regulatory agencies for inappropriate studies. We now have a much more robust database upon which to make certain conclusions and recommendations that were not dealt with extensively in S6. One of the questions to be considered is whether it is time to re-consider S6 and whether maintenance on this document is needed.
Increasing worldwide attention is being focused on a group of persistent organic pollutants known as the perfluorinated compounds (PFCs). This class of compounds includes perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) and a large number of other structurally related compounds sharing a variety of unique physical and chemical characteristics. Concern about these compounds is increasing as a growing number of studies indicate that they are toxic, bioaccumulative, and extraordinarily persistent in the environment. Moreover, the mean half-lives of human serum elimination of PFOS and PFOA have been estimated to be 5 and 4 years, respectively. While the vast majority of individuals living in the industrial world have measurable body burdens of the PFCs, almost nothing is known about how exposure to these compounds occurs. Very little is known about the potential health effects in humans, but a growing body of evidence indicates significant toxicity in various animal models. Given their unique chemical and physical properties, and their unusual persistence, these compounds are likely to be a major public health concern for the foreseeable future. This presentation summarizes information about the distribution of these materials in the environment, what is currently known about the possible routes of human exposure, and some of the key studies examining their toxicity.
The role of metabolism in the hepatotoxicity of the analgesic acetaminophen (APAP) is well described. At therapeutic doses the CYP metabolite N-acetyl-p-benzoquinone imine is detoxified by conjugation with glutathione (GSH); however, at high doses hepatic GSH is depleted and the metabolite covalently binds to protein. We and others have shown that covalent binding correlates with hepatic necrosis under a variety of conditions. However, biochemical mechanisms of toxicity are poorly understood. We have examined the role of mitochondrial permeability transition (MPT) and peroxynitrite in APAP toxicity in freshly isolated mouse hepatocytes. These hepatocytes have high levels of CYP enzymes necessary for development of toxicity. MPT occurs as a result of oxidative stress and leads to increased oxidative stress. It results in decreased ATP synthesis and is a lethal event. As previously reported we found that incubation of hepatocytes with APAP (1 mM) for 2 hrs results in minimal toxicity (ALT release). Subsequently hepatocytes were washed twice to remove APAP. Reincubation in media alone resulted in significant toxicity in the reincubation phase (3-5 hr) with toxicity occurring in approximately 75% of the hepatocytes by 5 hrs. The amount of toxicity was not significantly different if APAP was added back during the reincubation phase. Confocal microscopy studies utilizing the dye calcein indicated MPT.
Japan has long had a system of post-marketing safety surveillance including actual use studies (使用成績調査), special use studies (特定使用成績調査), expedited and periodic reporting of adverse drug reactions and in recent years this system has been augmented further by Early Postmarketing Pharmacovigilance (市販直後調査). Together these activities constitute a rigorous system of risk management. With the publication of the ICH E2E Guidance on Pharmacovigilance Plans and subsequent final guidances issued from the EU and FDA for Risk Management Plans and Risk Minimization Action Plans, there are now increased requirements outside Japan for companies to proactively identify both potential and confirmed risks based on an analysis of preclinical and clinical data and lay out a plan for monitoring, managing or reducing those risks. This presentation will review basic terms and concepts key to risk management systems from the CIOMS, ICH, FDA and EMEA guidance documents will be reviewed and areas where practices in the US and EU differ from those in Japan will be highlighted. The Risk Management Plan outlined by the ICH E2E and EU Guidances includes a Safety Specification which draws on preclinical and clinical experience and the role of toxicology and preclinical results in that plan will be described along with practical approaches to meeting the needs of these Specifications.
健常成人を対象としたいわゆる臨床第I相試験における第１の関心事は被験者の安全性である。とりわけヒトへの投与が初めてであるfirst in human (FIH)試験においてはきわめて注意深い試験計画の作成が必要である。これまでFIH試験は比較的安全に行われてきたが、その安全性への信頼をゆるがす事件が生じた。昨年３月のTGN1412事件である。免疫賦活作用を有する抗体であるTGN1412のFIH試験において、実薬投与例全例に重篤な有害反応が生じたこの事件は、全世界の臨床試験関係者に大きな衝撃を与えた。
この事件は大変な悲劇であり、安易な試験計画作成に対する警鐘であるが、必ずしも全ての化合物にあてはまるものではなく、FIH試験について極端に恐れなければならないものではない。これまで生物学的製剤を除いた低分子化合物については、非臨床試験に基づいたヒトでの安全性予測は充分に行われてきており、いわゆる第I相試験における重篤な有害事象の出現率はきわめて低い。MHRAと専門家による最終報告では、免疫に作用する薬物、生物学的製剤、新しい作用機序などの高リスクな化合物のFIH 試験についての提言が行われている。今回、この内容を紹介し、わが国におけるFIH 試験とそれをとりまく環境がどうあるべきかについて述べる。
ICH guidelines are available for all major steps of the non-clinical safety assessment of biologics, which includes therapeutic antibodies. In so far the development world could be simple – sponsors have just to follow the written recipes to bring their drugs through the process of non-clinical safety testing!? However, guidelines as well as international drug laws ask for scientifically sound testing as well as evaluation of drug candidates using state-of-the-art methods. Further, the regulatory practice and experience with other drugs under development may lead to additional requirements. All these emerging testing paradigms are considered essential to assure the highest safety standards for patients, but they also make preclinical safety scientists’ life interesting. Practice and challenges for non-clinical safety testing programs for antibodies under development in the US and in Europe will be presented and discussed.
Examples of recent experiences US companies gained during the regulatory process will be presented as well. Depending on its availability a guideline for the development of biologics announced by the US FDA may be discussed.
抗体医薬は、今や様々な領域において画期的な効果を発揮する新薬が登場し、コストの高さという難題はあるものの、従来の治療方法をドラスティックに進歩させるまでになったものまである。このため、あらたなアイデアの抗体医薬の開発は今後ますます活発になるものと思われる。そうした矢先に昨年３月に英国で発生したTGN1412によるFirst in Human Studyでの事故は世界中を揺るがした大事件であり、抗体医薬の発展に大きな影を落とすことになるのではないかと一時は深刻に危惧されたことは記憶に新しい。
Fertility is just one measure of reproductive function; parturition and lactation are other measures of female reproductive function. Standard fertility studies to evaluate toxicity to the female reproductive tract are designed to evaluate gamete maturation, mating, behavior, fertilization, reproductive function, pre-implantation development and implantation. Generally, female germ cell toxicity will be detected by endpoints measured in standard fertility study designs. If the studies incorporate oocyte quantification then the female germ cell as a target of toxicity can be ascertained. Most reproductive toxicants that have been shown to kill primordial or growing follicles are genotoxic, however, not all genotoxicants are ovarian follicle toxicants. Due to the complexity of normal female reproductive physiology, both in humans and standard rodent models, there are many different ways for xenobiotics to manifest reproductive toxicity. This myriad of potential mechanisms of toxicity is a primary reason why no defined hierarchy of sensitivity is applicable for the typical endpoint measured in standard fertility study designs. A weight-of-the-evidence approach incorporating the multiplicity of endpoints is the most prudent when determining a true impact on reproductive function. If a pattern of changes across endpoints in these study types are observed in a specific reproductive process where the mode of action is well understood and conserved across species, this should be considered a threat to human health unless data is available to the contrary.
GHS（Globally Harmonized System of Classification and Labeling of Chemicals）とは、国連が勧告している化学品の危険有害性の分類と表示に基づく情報伝達に関する世界的統一システムのことである。GHSでは、10項目の健康有害性について化学物質の有害性（ハザード）分類を行うが、その中には「生殖細胞変異原性」が含まれており、今や変異原性は、体細胞における「発がん関連影響」だけでなく、生殖細胞における「次世代への影響」にもあらためて焦点を当てつつある。GHSでは、ヒト生殖細胞における経世代突然変異誘発性に関する証拠の程度に応じた判定基準に基づき、生殖細胞変異原物質（GCM）を区分1A（既知ヒトGCM）、区分1B（ヒトGCMとみなすべきもの）および区分2（ヒトGCMの可能性があるもの）に有害性分類し、ラベルには、健康有害性シンボル、「危険」の注意喚起語、「遺伝性疾患のおそれ」等の有害性情報を表示する必要がある。混合物は、区分1のGCMが0.1%以上あるいは区分2のGCMが1.0%以上含まれる場合、それぞれ区分1あるいは区分2に分類する。判定基準の適合性に関しては、相反する知見の重み付けなど専門家判断が必要となる場合もある。一方、近年、GCMの検出には従来の“標準的”試験では十分ではない場合のあることが示されつつある。N-hydroxymethylacrylamideは、長期飲水投与優性致死試験においてのみ変異原性が確認され、生殖細胞特異的変異原物質の可能性が示唆されている。また、colchicineは、精母細胞より卵母細胞において顕著な異数性誘発がみられている。ヒトにおけるGCMのハザードおよびリスク評価には、性差や暴露期間・用量を考慮し、ヒト遺伝疫学の利用など、従来と異なるアプローチが必要と考えられる。