Clinical expert panel on monitoring potential lung toxicity of inhaled oligonucleotides: consensus points and recommendations

Oligonucleotides (ONs) are an emerging class of drugs being developed for the treatment of a wide variety of diseases including the treatment of respiratory diseases by the inhalation route. As a class, their toxicity on human lungs has not been fully characterized, and predictive toxicity biomarkers have not been identified. To that end, identification of sensitive methods and biomarkers that can detect toxicity in humans before any long term and/or irreversible side effects occur would be helpful. In light of the public's greater interests, the Inhalation Subcommittee of the Oligonucleotide Safety Working Group (OSWG) held expert panel discussions focusing on the potential toxicity of inhaled ONs and assessing the strengths and weaknesses of different monitoring techniques for use during the clinical evaluation of inhaled ON candidates. This white paper summarizes the key discussions and captures the panelists' perspectives and recommendations which, we propose, could be used as a framework to guide both industry and regulatory scientists in future clinical research to characterize and monitor the short and long term lung response to inhaled ONs.     

The position of renal denervation in treatment of hypertension: an expert consensus statement

Hypertension is an important risk factor for cardiovascular disease. In the Netherlands, there are approximately 2.8 million people with hypertension. Despite treatment recommendations including lifestyle changes and antihypertensive drugs, most patients do not meet guideline-recommended blood pressure (BP) targets. In order to improve BP control and lower the risk of subsequent cardiovascular events, renal sympathetic denervation (RDN) has been introduced and studied as a non-pharmacological approach. While early data on the efficacy of RDN showed conflicting results, improvements in treatment protocols and study design resulted in robust new evidence supporting the potential of the technology to improve patient care in hypertensive subjects. Recently, 5 randomised sham-controlled trials demonstrated the safety and efficacy of the technology. Modelling studies have further shown that RDN is cost-effective in the Dutch healthcare setting. Given the undisputable disease burden along with the shortcomings of current therapeutic options, we postulate a new, clearly framed indication for RDN as an adjunct in the treatment of hypertension. The present consensus statement summarises current guideline-recommended BP targets, proposed workup and treatment for hypertension, and position of RDN for those patients with primary hypertension who do not meet guideline-recommended BP targets (see central illustration).     

Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes.

Our laboratory has been routinely using suspended and cultured human hepatocytes for predicting drug metabolism and enzyme induction by drug candidates to aid drug discovery. Increasing limitation and irregular availability of human tissue has indicated the need for maximizing the use of this valuable resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by various methods has resulted in cells with poor metabolic activity and unacceptably low survival rates in culture. Recently, Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111-116) reported that cryopreserved rat hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37 degrees C in Krebs Ringer bicarbonate buffer prior to freezing. To further explore this methodology, both the functional capacity of the cells in culture as well as their ability to retain CYP inducibility were investigated with thawed cryopreserved hepatocytes. Although human hepatocytes were used in this study the initial work focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of cryopreserved hepatocytes, survival of the cells in culture was greatly enhanced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes were decreased to approximately 15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excellent plating efficiency (approximately 60%) and responded to classical CYP inducers dexamethasone, beta-naphthoflavone and phenobarbital in a manner indistinguishable from that of fresh hepatocytes. Experiments with human hepatocytes have also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture.     

Maintenance of steroid metabolism and hormone responsiveness in cryopreserved dog, monkey and human hepatocytes.

The efficient and effective use of hepatocytes from larger species and rare human material requires a reliable storage method for cells not needed on the day of preparation. Cryopreservation would seem to be the only viable alternative. In this study the suitability of a published cryopreservation technique on dog, monkey and human hepatocytes has been examined and the cells were tested for functionality directly after thawing and subsequent to culture using steroid metabolism and hormone responsiveness of glycogen phosphorylase a. Monkey and human hepatocytes appear to survive the freezing and thawing process better than dog cells-the latter losing the ability to respond to adrenergic stimuli and their ability to maintain steroid metabolism in culture. Although monkey and human cells do preserve their steroid metabolising capacity after freeze/thawing, there is not the significant increase in enzyme activity seen during culturing freshly isolated cells. It would appear, therefore, that some damage has occurred to the cells during the freeze/thaw process. As previously noted, Williams' medium E is superior to Ham's F-10 in maintaining enzyme activities in culture. It is suggested that cryopreservation is the way forward for the development of stockpiles of viable hepatocytes for biomedical and toxicological research and development but that further modifications to the process are still necessary to optimise the maintenance of liver-specific functions in the thawed cells.     

Optimal number of reagents required to evaluate hematolymphoid neoplasias: results of an international consensus meeting

At the ISAC 2000 Congress, the Clinical Cytometry Society organized a meeting of international experts to reach consensus on the minimum number of antibodies required for a full evaluation of hematologic and lymphoid neoplasias. A questionnaire was distributed prior to the meeting to numerous experts from US and European institutions and 13 responses were received. At the meeting, 25 individuals, including most of those who returned responses, participated in the discussions and voted on the issues presented. In chronic lymphoproliferative disorders (CLD), 9 antibodies (anti-CD5, CD19, kappa, lambda, CD3, CD20, CD23, CD10, and CD45) were deemed essential for initial evaluation by 75% of the participants. There was near unanimity that additional markers (selected from CD22, FMC7, CD11c, CD103, CD38, CD25, CD79b and heavy chains for B-cell disorders, and CD4, CD7, CD8, CD2, CD56, CD16, TCRa/b, and TCRg/d for T-cell disorders) would be needed to fully characterize CLD, although not every marker would be useful in all cases. Tissue lymphomas were believed to be similar to CLD, needing a minimum of 12--16 markers. However, for some cases, CD30, bcl-2, TdT, CD71, CD1a, and CD34 were cited as useful by the participants. Markers mentioned for plasma cell disorders included kappa, lambda, CD38, CD45, CD56, CD19, CD20, CD138, and heavy chains. Of 17 voting participants, 16 agreed that between 5 to 8 markers would be essential reagents for plasma cell disorders. For acute leukemia (AL), 10 markers (CD10, CD19, CD13, CD33, CD34, CD45, CD7, CD14, CD3, and HLADR) were considered essential by 75% of participants for initial characterization of the leukemia lineage. Most (>75%) agreed that at least one more B (CD20, CD22, CD79a, IgM), T (CD1a, CD2, CD4, CD5, CD8), myeloid (CD11b, CD15, CD64, CD117, myeloperoxidase), erythroid (CD36, CD71, glycophorin A), and megakaryocytic (CD41, CD61) reagents should be included in the essential panel. However, there was no agreement as to which was optimal. Thus, approximately 13--15 of those reagents would be considered essential in all cases of AL, whereas others (CD16, CD56, CDw65, TdT, and cytoplasmic CD3) were mentioned as useful in some cases. Almost all voting participants believed that the appropriate number of markers for complete characterization of AL would average 20--24. The majority of the responders (11 of 13) indicated that fewer reagents could be used in monitoring or staging patients with previously characterized disease, but not all ventured a specific number of reagents. From the above results, we conclude that the phenotypic analysis of hematologic and lymphoid neoplasia requires a rather extensive panel of reagents. Supplementary reagents might even be necessary if they prove to become relevant for diagnostic purposes. Reducing the number of antibodies could significantly compromise the diagnostic accuracy, appropriate monitoring, or therapy of these disorders.     

Early diagnosis of peripheral nervous system involvement in Fabry disease and treatment of neuropathic pain: the report of an expert panel

Background  

Fabry disease is an inherited metabolic disorder characterized by progressive lysosomal accumulation of lipids in a variety of cell types, including neural cells. Small, unmyelinated nerve fibers are particularly affected and small fiber peripheral neuropathy often clinically manifests at young age. Peripheral pain can be chronic and/or occur as provoked attacks of excruciating pain. Manifestations of dysfunction of small autonomic fibers may include, among others, impaired sweating, gastrointestinal dysmotility, and abnormal pain perception. Patients with Fabry disease often remain undiagnosed until severe complications involving the kidney, heart, peripheral nerves and/or brain have arisen.     

The structure of bacteriorhodopsin: an emerging consensus.

Six different sets of coordinates have been recently published for bacteriorhodopsin, with reported resolutions ranging from 3.5 A to 2. 3 A. Three of these are the result of electron crystallographic investigations of two-dimensional crystals of bacteriorhodopsin, whereas the others are from X-ray crystallographic studies of three-dimensional crystals of bacteriorhodopsin. How similar are these models? Are the structure determinations using X-ray diffraction data more accurate than those determined by electron crystallography? Is any one of these coordinate sets closer to the 'real' structure of bacteriorhodopsin than the others? Does the availability of newer models bring us closer to understanding how bacteriorhodopsin really works? These questions, as well as some related issues, are currently being explored.     

Standardized plant cell suspension test systems for an ecotoxicologic evaluation of the metabolic fate of xenobiotics

A rapid assay for the metabolic behaviour of organic chemicals in plants has been developed using soybean (Glycine max L.) and wheat (Triticum aestivum L.) cell suspension cultures. The test was performed with the following ¹⁴C-labelled compounds: 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloroaniline (4-CA), 3,4-dichloroaniline (3,4-DCA), pentachlorophenol (PCP), diethylhexylphthalate (DEHP), perylene (PR) and benzo[a]pyrene (BaP), which were applied at 1 mg · 1⁻¹ for 48 h during the logarithmic growth phase. All chemicals were catabolized The predominant fractions found were polar conjugates and non-extractable (bound) residues, with significantly less non-polar conversion products. Soybean cells often released large amounts of polar conjugates into the medium while in wheat cultures metabolites were mainly cell-associated. High rates of pentachlorophenol and chlorinated aniline incorporation into non-extractable residues were found in wheat suspension cells.     

The International Standard for Endotoxin: evaluation in an international collaborative study

An ampouled preparation of bacterial endotoxin, coded 84/650, was evaluated in 35 laboratories in 12 countries for its suitability to serve as the International Standard for Endotoxin. The ampouled preparation was calibrated in terms of the USA National Standard, EC5, in Limulus Amoebocyte Lysate gelation, turbidimetric and chromogenic tests and in rabbit pyrogen tests. On the basis of the results reported here, with the agreement of the participants in the study, and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 84/650 was established in 1986 as the International Standard for Endotoxin for Limulus Gelation Tests with an assigned unitage of 14,000 IU of endotoxin per ampoule.     

Cryopreservation and transplantation of hepatocytes: an approach for culture and clinical application

The development of a reproducible, effective system of cryopreservation of hepatocytes would create new possibilities for metabolic studies, as well as the clinical application of hepatocellular transplants in diverse hepatic disorders. For the purpose of dealing with both questions, we have studied different media and procedures of freezing, and have chosen that combination which afforded the best results for long-term studies of function. A series of intrasplenic transplants were performed with freshly isolated hepatocytes as well as cryopreserved ones, and several hepatospecific parameters (Alanine aminotransferase, ornithine carbamoyl transferase, lipogenesis, uptake of 99mTc-N-(p-butylphenylcarbamylmethyl)iminodiacetic acid, G6Pase, albumin, and gamma-glutamyl transpeptidase (gamma-GT) were quantified for 9 months. The histological study of the cells reveals that they acquired an architecture characteristic of the liver. All of the parameters indicative of hepatic function were detected throughout the 9-month period in the transplanted spleens. All the spleens, controls and transplanted, were found negative for the presence of the dedifferentiation marker gamma-GT. These results clearly indicate the possibility of recovering hepatocytes which maintain their specific functions after being subjected to a freezing-thawing process, with the corresponding implications for future clinical application as well as for hepatic biochemistry.     

Structural and functional evaluation of modifications in the composite skin graft: cryopreserved dermis and cultured keratinocytes.

Structural and functional aspects of modifications in the composite skin graft consisting of cultured keratinocytes and cryopreserved dermis were determined. Cryopreserved human cadaveric dermis separated from skin by short and mild trypsinization was compared with dermis obtained by prolonged incubation in medium and with fresh dermis obtained by the same methods. All types of dermis were shown to retain normal ultrastructure and topographic organization, as detected by scanning and transmission electron microscope and immunofluorescence analysis. However, in fresh skin, the layers were more firmly attached, mechanical separation was more difficult, and residual epidermis often remained attached to the dermis. Keratinocytes attached better, began replication earlier, and generally reached higher cell numbers when cultured on trypsinized dermis than on medium-treated dermis. The performance of several modifications in the reconstitution and grafting procedures of the composite skin graft after transplantation to athymic mice was examined. Cultured epidermis combined onto trypsinized or medium-treated whole and meshed dermis, dermis pregrafted and allowed to take before transplanting epidermis on top, and keratinocytes grown into multiple epithelia on top of trypsinized meshed or whole dermis prior to grafting. The best grafting results were obtained with an "instant" reconstituted skin model: multiple epithelia grown in vitro combined immediately before grafting onto meshed trypsinized dermis. The transplantation results of this modification were significantly better than those of all the other modifications, including initial growth of keratinocytes into multiple epithelia on top of trypsinized dermis prior to grafting.     

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.     

Risk assessment of gene flow from genetically engineered virus resistant cassava to wild relatives in Africa: an expert panel report

The probability and consequences of gene flow to wild relatives is typically considered in the environmental risk assessment of genetically engineered crops. This is a report from a discussion by a group of experts who used a problem formulation approach to consider existing information for risk assessment of gene flow from cassava (Manihot esculenta) genetically engineered for virus resistance to the ‘wild’ (naturalized) relative M. glaziovii in East Africa. Two environmental harms were considered in this case: (1) loss of genetic diversity in the germplasm pool, and (2) loss of valued species, ecosystem resources, or crop yield and quality due to weediness or invasiveness of wild relatives. Based on existing information, it was concluded that gene flow will occur, but it is not likely that this will reduce the genetic diversity in the germplasm pool. There is little existing information about the impact of the virus in natural populations that could be used to inform a prediction about whether virus resistance would lead to an increase in reproduction or survival, hence abundance of M. glaziovii. However, an increase in the abundance of M. glaziovii should be manageable, and would not necessarily lead to the identified environmental harms.     

稳定同位素在滨海湿地生态系统研究中的应用现状与前景

滨海湿地生态系统位于海陆交错地区,具有独特的生态系统特征、很高的服务功能和巨大的资源潜力,但同时也受到人为活动的严峻威胁.作为天然示踪物的稳定同位素为研究滨海湿地生态环境问题提供了重要手段.本文着重探讨了稳定同位素在滨海湿地动物食物源和食物网结构、滨海植物水分来源和利用效率、环境污染和生物入侵等全球变化对滨海湿地结构和功能的影响等方面的应用,指出了当前应用中存在的不足,如食物网研究中样品处理方式和富集度的确定,水分来源研究中水分抽提方法和仪器选择等,展望了稳定同位素在滨海湿地生态系统修复评价以及碳循环和温室气体排放研究中的应用前景.