Quanto costa validare un metodo alternativo al ricercatore che lo ha scoperto?

Come diventa obbligatorio un metodo alternativo?

Il ricercatore lo scopre, pubblica i risultati su una rivista scientifica, l’UE sceglie i metodi da validare, inizia l’iter di validazione con costi in buona parte a carico dell’azienda che propone il metodo, il metodo viene validato dopo 10 anni e viene inserito nelle linee guida in modo da diventare obbligatorio.

Quali sono le falle di questo sistema?

1. In primis, è l’Europa stessa a scegliere quali metodi siano da validare. Questa scelta aprioristica automaticamente costringe a scartare diverse potenziali metodologie alternative.
2. La validazione dura un tempo troppo lungo, spesso dunque i metodi validati non sono i più innovativi.
3. I risultati dei metodi alternativi, al posto di essere convalidati con dati conosciuti sull’uomo, vengono comparati con quelli dei test su animali. Considerate le limitatezze dell’animale, l’errore dello stesso può essere scambiato per un risultato giusto, mentre un risultato veramente esatto da parte del metodo alternativo può essere preso per un dato sbagliato (in quanto non concorde con quello dell’animale), conducendo talvolta allo scarto del metodo in fase di validazione.
   Un esempio è quello del Cytosensor Microphysiometer, che doveva rimpiazzare il Draize Test ma diede un risultato inizialmente inconcludente, dato che si confrontò il suo risultato con quello del Draize test stesso, finchè non fu validato grazie a meta-analisi retrospettive, che fecero capire che il problema non era nel metodo alternativo, ma nella presenza di falsi positivi che il Draize test tendeva a dare [1, 2].
4. Spesso non si validano prima i metodi applicabili in più campi, non si dà una priorità ai potenziali replacement nè alle metodiche che utilizzano tessuto umano e non animale.
5. L’Europa fornisce solo limitate risorse economiche per i costi della validazione, tali costi infatti includono la ripetizione di uno stesso esperimento in più laboratori. Una simile spesa dunque è sostenibile da un’azienda, mentre il singolo ricercatore normalmente non riuscirebbe, quindi spesso il paper in cui è descritto il metodo scoperto dal ricercatore va solo ad ingrossare le fila degli articoli di Pubmed, senza poter avere reali possibilità di venire un giorno impiegato come metodica obbligatoria in alternativa di un test animale.
6. Il tutto funziona in ambito regolatorio, mentre per la ricerca di base e applicata non esiste un database di metodi obbligatori costantemente aggiornato, è tutto delegato all’abilità del ricercatore di trovare quello che egli reputa il metodo appropriato di sostituzione (senza che per forza lo sia), se ritiene che vi sia un metodo alternativo.

Per quanto riguarda il punto 5, questa spesa a quanto ammonta? Secondo la dottoressa Meg Lewis dell’azienda biotech “Kirkstall Ltd” (che si occupa di metodi alternativi), come riportato da un paper di ALTEX del 2013, a 5 milioni di Euro per ogni nuovo metodo da validare.

Non è dunque il caso che l’Europa si prenda carico interamente del costo di validazione dei metodi alternativi, e che smetta di limitare l’ingresso all’iter di validazione a metodi scelti aprioristicamente?

Riportiamo l’articolo da cui è preso il dato [3]:

“In the EU, and indeed most other developed countries, the testing of new clinical entities (NCEs) on animals is required by law in order to determine the safety of a new drug before it can be tested on humans. However, the effectiveness of this process is much disputed, given the frequency with which animal testing fails to accurately predict the reactions and interactions of a drug in humans, leading to toxic insult and even death in patients. There are perhaps two explanations for this failure rate – firstly, that the current paradigm of drug discovery is flawed, and secondly that the methodologies employed as part of that paradigm are insufficient to determine the safety and efficacy of new drugs. With each new drug costing an average of $ 6bn to get to market, and with NCE output decreasing annually despite annual R&D spend increases, the pharmaceuticals and CROs are beginning to recognize the limits in cost and effectiveness of current drug discovery approaches.

Alternative methods to traditional testing are required. New EU legislation (Directive 2010/63/EU) states that, where there is an alternative to animal experimentation, that alternative must be used. These methods are on or near to market, and promise to deliver more accurate and sensitive tests for toxicity than animal testing, but early widespread adoption of these new technologies is delayed by the structure of the validation strategy – with an estimated investment of 10 years and € 5m for ECVAM validation of a new assay for example, change is not likely to happen quickly.

If not ethical concern over animal testing, the economic climate is likely to be a key motivating factor in helping drug companies move away from traditional methods and towards new alternatives to current practices, because animal testing is expensive in addition to its limited effectiveness. Alternative approaches promise to reduce the cost of pre-clinical screening, as well as reducing and eventually replacing animal use. Advances in in vitro cell culture technology, combined with quantitative systems pharmacology and new computational approaches to drug discovery, will pave the way for a future of cheaper and more accurate compound testing, better hit rates, and safer and more effective drugs.”

La stessa dottoressa Lewis aveva già riferito in un’intervista [4]:

La validazione rimane un intralcio significativo per l’implementazione di metodi alternativi da parte dell’industria. Con 10 anni e 5 milioni di euro di investimento medio per ottenere un singolo test validato dall’ECVAM, il costo è semplicemente troppo grande per la maggior parte delle piccole imprese, ma senza di essa, è molto difficile convincere le organizzazioni di ricerca a contratto e farmaceutiche ad utilizzare le nuove tecnologie e cambiare i loro approcci (se non impossibile).

Riferimenti bibliografici:

1. [Hartung T, Bruner L, Curren R, Eskes C, Goldberg A, McNamee P, Scott L, Zuang V. First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined applicability domain. ALTEX. 2010;27(1):43-51.]

2. [Goldberg AM, Hartung T. Protecting more than animals. Sci Am. 2006 Jan;294(1):84-91.].

3. [M. Lewis, J.M. Wilkinson. Moving forward: a new paradigm for drug discovery. ALTEX Proceedings 2, 2/13, LINZ 2013, p. 68.]

4. https://vivoveritas.wordpress.com/2013/09/05/intervista-con-la-drssa-meg-lewis-della-kirkstall-ltd-sul-sistema-quasi-vivo/

Metodi in vitro di barriera epiteliale (pelle, polmoni e intestino) in tossicologia e farmacologia

[Gordon S, Daneshian M, Bouwstra J, Caloni F, Constant S, Davies DE, Dandekar G, Guzman CA, Fabian E, Haltner E, Hartung T, Hasiwa N, Hayden P, Kandarova H, Khare S, Krug HF, Kneuer C, Leist M, Lian G, Marx U, Metzger M, Ott K, Prieto P, Roberts MS, Roggen EL, Tralau T, van den Braak C, Walles H, Lehr CM. Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology. ALTEX. 2015;32(4):327-78.]

Abstract:

“Models of the outer epithelia of the human body – namely the skin, the intestine and the lung – have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.”

Tox21 su Nature: metodi in vitro per sostituire gli animali negli studi di tossicità

[Huang R, Xia M, Sakamuru S, Zhao J, Shahane SA, Attene-Ramos M, Zhao T, Austin CP, Simeonov A. Modelling the Tox21 10 K chemical profiles for in vivo toxicity prediction and mechanism characterization. Nat Commun. 2016 Jan 26;7:10425.]

Abstract:

Target-specific, mechanism-oriented in vitro assays post a promising alternative to traditional animal toxicology studies. Here we report the first comprehensive analysis of the Tox21 effort, a large-scale in vitro toxicity screening of chemicals. We test ~10,000 chemicals in triplicates at 15 concentrations against a panel of nuclear receptor and stress response pathway assays, producing more than 50 million data points. Compound clustering by structure similarity and activity profile similarity across the assays reveals structure–activity relationships that are useful for the generation of mechanistic hypotheses. We apply structural information and activity data to build predictive models for 72 in vivo toxicity end points using a cluster-based approach. Models based on in vitro assay data perform better in predicting human toxicity end points than animal toxicity, while a combination of structural and activity data results in better models than using structure or activity data alone. Our results suggest that in vitro activity profiles can be applied as signatures of compound mechanism of toxicity and used in prioritization for more in-depth toxicological testing.

Mancanza di concordanza nel rilevamento dei non-carcinogeni tra ratti e topi e tra maschi e femmine

[Wang B, Gray G. Concordance of Noncarcinogenic Endpoints in Rodent Chemical Bioassays. Risk Anal. 2015 Jun;35(6):1154-66.]

Abstract:

“Prediction of noncancer toxicologic outcomes in rodent bioassays of 37 chemicals from the National Toxicology Program was evaluated. Using the nonneoplastic lesions noted by NTP pathologists, we evaluate both agreement in toxic lesions across experiments and the predictive value of the presence (or absence) of a lesion in one group for other groups. We compare lesions between mice and rats, male mice and male rats, and female mice and female rats in both short-term and long-term bioassays. We also examine whether lesions found in a specific organ in a short-term test are also found in the long-term test of the same chemical. We find agreement (concordance) across species for specific lesions, as evaluated by the Kappa statistic, ranging from 0.58 (for concordance of nasal lesions between female mice and rats in long-term studies) to -0.14 (lung lesions between mice and rats in long-term studies). Predictive values are limited by the relatively small numbers of observations of each type of lesion. Positive predictive values range from 100% to 0%. Comparing the lesions found in short-term tests to those found in long-term tests resulted in Kappa statistic values from 0.76 (spleen lesions in male rats) to -0.61 (lung lesions in female mice). Positive predictive values of short-term tests for long-term tests range from 70% to 0%. Overall, there is considerable uncertainty in predicting the site of toxic lesions in different species exposed to the same chemical and from short-term to long-term tests of the same chemical.”

Differenze tra geni ortologhi nell’uomo e nel topo

[Walid H. Gharib and Marc Robinson-Rechavi. When orthologs diverge between human and mouse. Brief Bioinform. 2011 Sep; 12(5): 436–441.]

Abstract:

Despite the common assumption that orthologs usually share the same function, there have been various reports of divergence between orthologs, even among species as close as mammals. The comparison of mouse and human is of special interest, because mouse is often used as a model organism to understand human biology. We review the literature on evidence for divergence between human and mouse orthologous genes, and discuss it in the context of biomedical research.

Nel testo:

We believe that both small-scale and large-scale studies provide evidence that functional divergence between human and mouse orthologs, although a minority phenomenon, still affects a significant proportion of genes. Divergence of gene expression, of alternative splicing, and of mutant phenotypes, each affect of the order of 10–20% of ortholog pairs, under conservative estimates. If these and other different processes affect different genes, then it might be a majority of genes which are affected. But even if the same genes differ in expression pattern, splicing, etc., then having ~15% of human-mouse orthologs with strong differences will affect many pathways and biological processes of interest.

Cellule staminali paziente-specifiche per studiare epilessia, sindrome di Rett, di Timothy, di Phelan-McDermid e di Dravet

[Tidball AM, Parent JM. Exciting Cells: Modeling Genetic Epilepsies with Patient-Derived Induced Pluripotent Stem Cells. Stem Cells. 2015 Sep 7. doi: 10.1002/stem.2203.] 

Abstract:

Human induced pluripotent stem cell (iPSC) models of epilepsy are becoming a revolutionary platform for mechanistic studies and drug discovery. The skyrocketing pace of epilepsy gene discovery is vastly outstripping the development of in vivo animal models. Currently, antiepileptic drug prescribing to patients with specific genetic epilepsies is based on small-scale clinical trials and empiricism; however, rapid production of patient-derived iPSC models will allow for precision therapy. We review iPSC-based studies that have already afforded novel discoveries in diseases with epileptic phenotypes, as well as challenges to using iPSC-based neurological disease models. We also discuss iPSC-derived cardiomyocyte studies of arrhythmia-inducing ion channelopathies that exemplify novel drug discovery and use of multielectrode array technology that can be translated to epilepsy research. Beyond initial studies of Rett, Timothy, Phelan-McDermid, and Dravet syndromes, the stage is set for groundbreaking iPSC-based mechanistic and therapeutic discoveries in genetic epilepsies with the potential to impact patient treatment and quality of life.

I test di tossicità in vitro e le analisi genomiche sono più accurate degli studi su animali in questo campo: il caso del tabacco

[Manuppello JR, Sullivan KM. Toxicity assessment of tobacco products in vitro. Altern Lab Anim. 2015 Mar;43(1):39-67.]

Abstract:

Driven by new regulatory demands to demonstrate risk reduction, the toxicity assessment of tobacco products increasingly employs innovative in vitro methods, including biphasic cell and tissue cultures exposed to whole cigarette smoke at the air-liquid interface, cell transformation assays, and genomic analyses. At the same time, novel tobacco products are increasingly compared to traditional cigarettes. This overview of in vitro toxicology studies of tobacco products reported in the last five years provides evidence to support the prioritisation of in vitro over in vivo methods by industry and their recommendation by regulatory authorities.

Nel testo:

“Combining data from human cells and tissues with existing data from human epidemiology and clinical studies, may provide insights into potential harm reduction strategies, while avoiding the extrapolation issues often associated with in vivo animal studies.
In general, studies that use in vitro methods have the ability to assess potential MRTPs more quickly and to provide more-specific, actionable and human relevant data than do animal studies. In vitro models can also better reflect genetic and environmental differences within the human population (54), which can be important for tobacco addiction (55) and toxicity (56) studies“.

 

Colture neuronali da cellule staminali in ambito farmacologico e tossicologico

[Smith I, Silveirinha V, Stein JL, de la Torre-Ubieta L, Farrimond JA, Williamson EM, Whalley BJ. Human neural stem cell-derived cultures in three-dimensional substrates form spontaneously functional neuronal networks. J Tissue Eng Regen Med. 2015 Feb 25.]

Abstract:

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

 

I difetti e i danni all’Uomo della sperimentazione animale

[Akhtar A. The flaws and human harms of animal experimentation. Camb Q Healthc Ethics. 2015 Oct;24(4):407-19.]

Full Text: http://journals.cambridge.org/action/displayFulltext?type=6&fid=9949938&jid=CQH&volumeId=24&issueId=04&aid=9949937&bodyId=&membershipNumber=&societyETOCSession=&fulltextType=RA&fileId=S0963180115000079

Abstract:

Nonhuman animal (“animal”) experimentation is typically defended by arguments that it is reliable, that animals provide sufficiently good models of human biology and diseases to yield relevant information, and that, consequently, its use provides major human health benefits. I demonstrate that a growing body of scientific literature critically assessing the validity of animal experimentation generally (and animal modeling specifically) raises important concerns about its reliability and predictive value for human outcomes and for understanding human physiology. The unreliability of animal experimentation across a wide range of areas undermines scientific arguments in favor of the practice. Additionally, I show how animal experimentation often significantly harms humans through misleading safety studies, potential abandonment of effective therapeutics, and direction of resources away from more effective testing methods. The resulting evidence suggests that the collective harms and costs to humans from animal experimentation outweigh potential benefits and that resources would be better invested in developing human-based testing methods.

Nel testo:

Wide differences have also become apparent in the regulation of the same genes, a point that is readily seen when observing differences between human and mouse livers. ⁴⁸ Consistent phenotypes (observable physical or biochemical characteristics) are rarely obtained by modification of the same gene, even among different strains of mice. ⁴⁹ Gene regulation can substantially differ among species and may be as important as the presence or absence of a specific gene. Despite the high degree of genome conservation, there are critical differences in the order and function of genes among species. To use an analogy: as pianos have the same keys, humans and other animals share (largely) the same genes. Where we mostly differ is in the way the genes or keys are expressed. For example, if we play the keys in a certain order, we hear Chopin; in a different order, we hear Ray Charles; and in yet a different order, it’s Jerry Lee Lewis. In other words, the same keys or genes are expressed, but their different orders result in markedly different outcomes.

Recognizing the inherent genetic differences among species as a barrier to translation, researches have expressed considerable enthusiasm for genetically modified (GM) animals, including transgenic mice models, wherein human genes are inserted into the mouse genome. However, if a human gene is expressed in mice, it will likely function differently from the way it functions in humans, being affected by physiological mechanisms that are unique in mice. For example, a crucial protein that controls blood sugar in humans is missing in mice. ⁵⁰ When the human gene that makes this protein was expressed in genetically altered mice, it had the opposite effect from that in humans: it caused loss of blood sugar control in mice. Use of GM mice has failed to successfully model human diseases and to translate into clinical benefit across many disease categories. ⁵¹ Perhaps the primary reason why GM animals are unlikely to be much more successful than other animal models in translational medicine is the fact that the “humanized” or altered genes are still in nonhuman animals.

In many instances, nonhuman primates (NHPs) are used instead of mice or other animals, with the expectation that NHPs will better mimic human results. However, there have been sufficient failures in translation to undermine this optimism. For example, NHP models have failed to reproduce key features of Parkinson’s disease, both in function and in pathology. ⁵² Several therapies that appeared promising in both NHPs and rat models of Parkinson’s disease showed disappointing results in humans.⁵³ The campaign to prescribe hormone replacement therapy (HRT) in millions of women to prevent cardiovascular disease was based in large part on experiments on NHPs. HRT is now known to increase the risk of these diseases in women. ⁵⁴

e:

“Appreciation of differences” and “caution” about extrapolating results from animals to humans are now almost universally recommended. But, in practice, how does one take into account differences in drug metabolism, genetics, expression of diseases, anatomy, influences of laboratory environments, and species- and strain-specific physiologic mechanisms—and, in view of these differences, discern what is applicable to humans and what is not? If we cannot determine which physiological mechanisms in which species and strains of species are applicable to humans (even setting aside the complicating factors of different caging systems and types of flooring), the usefulness of the experiments must be questioned.

It has been argued that some information obtained from animal experiments is better than no information. ⁶⁴ This thesis neglects how misleading information can be worse than no information from animal tests. The use of nonpredictive animal experiments can cause human suffering in at least two ways: (1) by producing misleading safety and efficacy data and (2) by causing potential abandonment of useful medical treatments and misdirecting resources away from more effective testing methods.

Humans are harmed because of misleading animal testing results. Imprecise results from animal experiments may result in clinical trials of biologically faulty or even harmful substances, thereby exposing patients to unnecessary risk and wasting scarce research resources. ⁶⁵ Animal toxicity studies are poor predictors of toxic effects of drugs in humans. ⁶⁶ As seen in some of the preceding examples (in particular, stroke, HRT, and TGN1412), humans have been significantly harmed because investigators were misled by the safety and efficacy profile of a new drug based on animal experiments. ⁶⁷ Clinical trial volunteers are thus provided with raised hopes and a false sense of security because of a misguided confidence in efficacy and safety testing using animals.

An equal if indirect source of human suffering is the opportunity cost of abandoning promising drugs because of misleading animal tests. ⁶⁸ As candidate drugs generally proceed down the development pipeline and to human testing based largely on successful results in animals ⁶⁹ (i.e., positive efficacy and negative adverse effects), drugs are sometimes not further developed due to unsuccessful results in animals (i.e., negative efficacy and/or positive adverse effects). Because much pharmaceutical company preclinical data are proprietary and thus publicly unavailable, it is difficult to know the number of missed opportunities due to misleading animal experiments. However, of every 5,000–10,000 potential drugs investigated, only about 5 proceed to Phase 1 clinical trials. ⁷⁰ Potential therapeutics may be abandoned because of results in animal tests that do not apply to humans. ⁷¹ Treatments that fail to work or show some adverse effect in animals because of species-specific influences may be abandoned in preclinical testing even if they may have proved effective and safe in humans if allowed to continue through the drug development pipeline.

Modello “on a chip” di barriera placentare

[Lee JS, Romero R, Han YM, Kim HC, Kim CJ, Hong JS, Huh D. Placenta-on-a-chip: a novel platform to study the biology of the human placenta. J Matern Fetal Neonatal Med. 2015 Jun 15:1-9.]

Full Text: http://informahealthcare.com/doi/full/10.3109/14767058.2015.1038518

Abstract:

“Objective: Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta.

Materials and methods: A “Placenta-on-a-Chip” microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone.

Results: Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations.

Conclusion: The “Placenta-on-a-Chip” microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.”