Archivi del mese: dicembre 2013

Dobbiamo sostituire la sperimentazione animale: questo è sotto gli occhi di tutti

Andiamo oggi a commentare un articolo pro-sperimentazione animale apparso sul Quotidiano Sanità, “Sperimentazione animale. I risultati? Davanti ai nostri occhi”.

All’inizio, l’autore, per dimostrare la validità della SA, cita diversi esempi di scoperte che hanno avuto origine grazie a ricerche su animali.

Questa strategia, ovviamente, è fallace, si tratta infatti di dati aneddotici che non ci permettono di valutare efficacemente l’animale, per farlo servirebbe comparare il numero di successi assieme a quello dei fallimenti e poi trarne le conclusioni.

Egli, inoltre, commenta alcuni studi:

– Seok et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. PNAS 26 febr 2013 vol 110 n°9 3507-3512.
– Hackam & Redelmeier. Translation of research evidence from animals to humans. JAMA 2006;296(14):1731-2.
– Pound et al. Where is the evidence that animal research benefits humans? British BMJ. 2004 February 28; 328(7438): 514–517.
– van Meer PJ, Kooijman M, Gispen-de Wied CC, Moors EH, Schellekens H. The ability of animal studies to detect serious post marketing adverse events is limited. Regul Toxicol Pharmacol. 2012 Dec;64(3):345-9.
– Van der Worp et al. 2011. Preclinical studies of human disease: time to take methodological quality seriously. J Mol Cell Cardiol. 2011 Oct;51(4):449-50.

Afferma, sul primo:

“lo studio sui modelli animali per le malattie infiammatorie riguarda in realtà un solo modello animale, il topoC57BL/6J;”

In realtà questo studio è stato solo uno dei molti fallimenti del modello animale, che non fa altro che aggiungersi agli altri: infatti, “the paper by Seok is not a stand-alone study, but it was triggered by worrying findings of 20 years of research, which suggested that non-predictive animal models might be the reason for the many clinical failures of new drugs in the field of sepsis.” e “back to stroke: how well do the animal models work? They work similar as in inflammation: not at all.” [1]
In definitiva, analizzando l’animale da una prospettiva globale, esso si rivela estremamente limitato e a più riprese fallimentare.

Passa dunque al secondo:

“Lo studio stesso di Hackman su JAMA parla di scarsa qualità di come vengono impostati certi studi sul modello animale e non critica lo stesso”

In primis è Hackam, e non Hackman.
In secondo luogo non è vero ciò che si dice, dato che il paper prende in considerazione proprio gli studi di alta qualità.
Infatti, gli autori del paper affermano:
“Finally, poor replication of even high-quality animal studies should be expected by those who conduct clinical research.”

“prende in considerazione un numero limitato di studi (76) in un arco di tempo limitato (20 anni, dal 1980 al 2000); decisamente pochi e decisamente un arco di tempo ridotto per vedere le conseguenze di uno studio in ambito clinico.”

In realtà anche articoli precedenti prendono in considerazione un numero simile di studi, e in un periodo ancora più breve, come Ioannidis JPA. Contradicted and initially stronger effects in highly cited clinical research. JAMA. 2005;294:218-228., dando addirittura una stima di successo maggiore.

Considerato dunque che Hackam prende in considerazione praticamente il doppio del tempo, facendo abbassare la soglia di successo dal 44% al 33%, si potrebbe pensare che un tempo maggiore potrebbe rivelare una stima di successo dell’animale ancora minore.

In aggiunta, Grant e colleghi hanno concluso che le ricerche di base necessitano di un periodo di circa 17 anni per avere un impatto in fase clinica, quindi il tempo preso in considerazione da Hackam e Redelmeier è adeguato [2] [3].

“lo studio di Pound sul BMJ non è esente da critiche in quanto tende a prendere ad esempio solo determinati risultati e si dimentica di riportare anche frasi come “There were no differences between the results of the animal experiments and clinical studies” presente nello studio sulla Nimodipina o “In fact, there were no differences between the results of these experiments (animals) and clinical trials” nel lavoro di Lucas sulla laserterapia.”

In realtà le citazioni dello studio della nimodipina e della laserterapia non erano collegate a una valutazione delle stesse, nel lavoro di Pound, semmai erano prove di situazioni in cui i trial clinici non erano stati preceduti dagli esperimenti su animali, ma erano stati eseguiti in contemporanea, così come afferma lo stesso articolo, nella parte delle implicazioni:

“Le prove cliniche della nimodipina e della terapia laser di basso livello sono state condotte contemporaneamente agli studi su animali, mentre quelle sulla rianimazione con fluidi, sulla terapia trombolitica e sugli antagonisti dell’endotelina sono state intraprese malgrado fossero disponibili prove di effetti dannosi sugli animali. Ciò indica che i dati derivanti dagli animali sono stati considerati non pertinenti, il che fa sorgere dubbi sul motivo che aveva spinto a intraprenderli e mina alla base il principio che gli esperimenti sugli animali siano necessari allo sviluppo della medicina umana.”

In parole povere, non si valuta l’esito di queste ricerche, ma si fa notare come venga percepita dal ricercatore stesso il risultato su animali come qualcosa di non pertinente, di non necessario.
Ovviamente, per valutare l’effettiva utilità della SA, non possiamo prendere singoli casi fuori dai loro contesti, ma dobbiamo valutarli in maniera globale, pertanto l’affermazione di critica all’articolo di Pound non significa nulla.

“lo studio di Van Meera […] elogia il modello animale per quanto riguarda lo studio delle dosi terapeutiche e gli effetti legati alla farmacodinamica, ossia al meccanismo d’azione del farmaco”

Dove sarebbe scritto questo? Soprattutto la farmacodinamica nell’intero articolo non viene mai citata, se non una volta, senza alcuna valutazione del modello, nella sezione “Metodi”:

“Primary and secondary pharmacodynamic data, safety pharmacology data, and single and repeat dose toxicology data were reviewed to identify in vivo events in any rodent or non-rodent species and at any dose or time point that could be considered to be associated with the SAR described in the Product Safety Announcement or Direct Healthcare Practitioner Communications.”

Il meccanismo d’azione viene citato solo una volta e non in relazione agli animali, quindi dove lo loderebbe?

A noi sembra invece che il paper affermi la fallacia della SA, infatti dice:

“We showed that the animal studies performed to evaluate the safety of new small molecule drugs are not sensitive enough to predict post-marketing SARs. Therefore, it is not relevant to include animal study data for prospective pharmacovigilance studies.”

Inoltre, accenna addirittura alla necessità di superare l’animale al più presto:

“From this, possibilities based on scientific facts may develop which allow new technologies to be implemented that predict the safety and efficacy of therapeutics equal to or better than animal studies do.”

“il lavoro di Van der Woop non critica il modello animale in se’ ma la ridotta capacità traslazionale di studi sul modello animale in ambito cardiologico (in particolare si concentra sugli infarti) a causa di bias quali la scarsa qualità di alcuni studi, la non riproducibilità di alcune condizioni importanti nella pratica clinica (per esempio l’utilizzo di topi “giovani” per pat
ologie che interessano soggetti anziani), etc; si criticano quindi le modalità con cui vengono fatti gli esperimenti e non l’uso dell’animale stesso;”

D’altra parte lo stesso autore, in un altro articolo, oltre a criticare la qualità metodologica degli studi su animali, afferma che, anche con eventuali miglioramenti, il risultato del modello animale non risulterebbe comunque soddisfacente, ma avrebbe una traslazione di appena una volta su 3:

“In a review of animal studies published in seven leading scientific journals of high impact, about one-third of the studies translated at the level of human randomised trials, and one-tenth of the interventions, were subsequently approved for use in patients [1]. However, these were studies of high impact (median citation count, 889), and less frequently cited animal research probably has a lower likelihood of translation to the clinic. Depending on one’s perspective, this attrition rate of 90% may be viewed as either a failure or as a success, but it serves to illustrate the magnitude of the difficulties in translation that beset even findings of high impact” [4].

Bibliografia:

[1] Leist M, Hartung T. Inflammatory findings on species extrapolations: humans are definitely no 70-kg mice. Arch Toxicol. 2013 Apr;87(4):563-7.

[2] Grant J, Cottrell R, Cluzeau F, Fawcett G. Evaluating “payback” on biomedical research from papers cited in clinical guidelines: applied bibliometric study. BMJ 2000, 320(7242):1107-1111.

[3] Grant J, Green L, Mason B. Basic research and health: a reassessment of the scientific basis for the support of biomedical science. Research Evaluation 2003, 12(3):217-224.

[4] Van der Worp, H. B., Howells, D. W., Sena, e. S., et al. (2010). Can animal models of disease reliably inform human studies? PLoS Med 7, e1000245.

Annunci

Predizione del metabolismo tessuto-specifico umano

[Shlomi T, Cabili MN, Herrgård MJ, Palsson BØ, Ruppin E. Network-based prediction of human tissue-specific metabolism. Nat Biotechnol. 2008 Sep;26(9):1003-10. doi: 10.1038/nbt.1487.]

Full Text: http://llama.mshri.on.ca/courses/Biophysics205/Papers/Shlomi_2008.pdf

Abstract:

L’indagine diretta in vivo del metabolismo dei mammiferi è complicata dalle diverse funzioni metaboliche dei diversi tessuti. Presentiamo un metodo computazionale che descrive correttamente la specificità tissutale del metabolismo umano su larga scala. Integrando i dati tessuto specifici di espressione genica e proteica con un’esistente comprensiva ricostruzione della rete metabolica globale umana, prevediamo l’attività metabolica tessuto-specifica in dieci tessuti umani. Questo rivela un ruolo centrale per la regolazione post-trascrizionale nel plasmare profili di attività metabolica tessuto-specifici. La specificità tissutale predetta dei geni responsabili di malattie metaboliche e di differenze tessuto-specifiche nello scambio di metaboliti con biofluidi estendono notevolmente al di là delle differenze tessuto-specifiche manifestate nei dati di espressione enzimatica, e sono validati dall’estrazione a larga scala di dati di tessuto-specificità. I nostri risultati stabiliscono una base computazionale per lo studio dell’intero genoma del metabolismo umano normale e anormale in una maniera tessuto-specifica.

Integrated Testing Strategy (ITS) e nuove tecnologie nella sostituzione dei test su animali per la valutazione del rischio di sostanze chimiche, cosmetici e farmaci

[Leist M, Lidbury BA, Yang C, Hayden PJ, Kelm JM, Ringeissen S, Detroyer A, Meunier JR, Rathman JF, Jackson GR Jr, Stolper G, Hasiwa N. Novel technologies and an overall strategy to allow hazard assessment and risk prediction of chemicals, cosmetics, and drugs with animal-free methods. ALTEX. 2012;29(4):373-88.]

Full Text: http://www.altex.ch/resources/raltex_2012_4_373_388_Leist11.pdf

Abstract:

Several alternative methods to replace animal experiments have been accepted by legal bodies. An even larger number of tests are under development or already in use for non-regulatory applications or for the generation of information stored in proprietary knowledge bases. The next step for the use of the different in vitro methods is their combination into integrated testing strategies (ITS) to get closer to the overall goal of predictive “in vitro-based risk evaluation processes.” We introduce here a conceptual framework as the basis for future ITS and their use for risk evaluation without animal experiments. The framework allows incorporation of both individual tests and already integrated approaches. Illustrative examples for elements to be incorporated are drawn from the session “Innovative technologies” at the 8th World Congress on Alternatives and Animal Use in the Life Sciences, held in Montreal, 2011. For instance, LUHMES cells (conditionally immortalized human neurons) were presented as an example for a 2D cell system. The novel 3D platform developed by InSphero was chosen as an example for the design and use of scaffold-free, organotypic microtissues.The identification of critical pathways of toxicity (PoT) may be facilitated by approaches exemplified by the MatTek 3D model for human epithelial tissues with engineered toxicological reporter functions. The important role of in silico methods and of modeling based on various pre-existing data is demonstrated by Altamira’s comprehensive approach to predicting a molecule’s potential for skin irritancy. A final example demonstrates how natural variation in human genetics may be overcome using data analytic (pattern recognition) techniques borrowed from computer science and statistics. The overall hazard and risk assessment strategy integrating these different examples has been compiled in a graphical work flow.

Conclusioni:

Each of the technical approaches and model systems presented here has been developed as a stand-alone method. Most have been developed for a specific purpose and to solve defined problems. Dozens, if not hundreds, of such technologies are already available, and only a few have been picked to exemplify the progress in the field. We have put forward the hypothesis here that added value may be generated by a combination of such approaches. The approach taken here may, at first glance, look different from or in competition with other new strategies. For instance, the ToxCast program, or different approaches that follow the “Tox21″ vision take a different starting point. In their extreme form, they rid themselves of the old patchwork of different toxicological models, be they in vivo or in vitro, and put forward a new homogeneous framework, based, for instance, on PoTand systems biology modeling. It is not yet clear, which role assays play that use endpoints that are toxicologically apparently simple but (systems-) biologically highly complex, e.g., cell death, neurite degeneration, or albumin secretion. Here we take an alternative approach to define an overall scaffold of what information would contribute to an animal-free risk assessment. This scaffold is used to recruit a largely heterogeneous group of assays, providing information at different levels of complexity, with different throughput rates, and possibly with different information value. Combined in a scheme, these assays can fill knowledge gaps and improve the overall risk assessment of chemicals for which little is known. The framework suggested here is also suited to the incorporation of individual tests and in silico methods developed for Tox21, or even to incorporation of testing strategies at a higher level of integration, as shown by the Altamira example of skin irritancy modeling. Thus, this approach may represent a practical solution for high production volume risk assessment in the intermediate future, while many tests are still under development and no complete test platform on the basis of PoTtesting is available. The future will then bring higher throughput assays, better systems biology modeling, better integration of data from omics technologies, and better cell sources. For instance, we envisage that testing in non-transformed cell models, of murine or preferentially of human origin, will require a further development of stem cell technology, to provide reliable cell sources.

Ingegneria tissutale nella sostituzione della sperimentazione animale

[Holmes A, Brown R, Shakesheff K. Engineering tissue alternatives to animals: applying tissue engineering to basic research and safety testing. Regen Med. 2009 Jul;4(4):579-92. doi: 10.2217/rme.09.26.]

Abstract:

The focus for the rapid progress in the field of tissue engineering has been the clinical potential of the technology to repair, replace, maintain or enhance the function of a particular tissue or organ. However, tissue engineering has much wider applicability in basic research and safety testing, which is often not recognized owing to the clinical focus of tissue engineers. Using examples from a recent National Centre for the Replacement, Refinement and Reduction of Animals in Research/Biotechnology and Biological Sciences Research Council symposium, which brought together tissue engineers and scientists from other research communities, this review highlights the potential of tissue engineering to provide scientifically robust alternatives to animals to address basic research questions and improve drug and chemical development in the pharmaceutical and chemical industries.

[BéruBé K, Gibson C, Job C, Prytherch Z. Human lung tissue engineering: a critical tool for safer medicines. Cell Tissue Bank. 2011 Feb;12(1):11-3. doi: 10.1007/s10561-010-9204-6. Epub 2010 Sep 8.] 

Abstract:

In the field of human tissue-engineering, there has been a strong focus on the clinical aspects of the technology, i.e. repair, replace and enhance a given tissue/organ. However, much wider applications for tissue engineering (TE) exist outside of the clinic that are often not recognised, and include engineering more relevant models than animals in basic research and safety testing. Traditionally, research is initially conducted on animals or cell lines, both of which have their limitations. With regard to cell lines, they are usually transformed to enable indefinite proliferation. These immortalised cell lines provide the researcher with an almost limitless source of material. However, the pertinence of the data produced is now under scrutiny, with the suggestion that some historical cell lines may not be the cell type originally reported. By engineering normal, biomimetic (i.e. life-mimicking), human tissues with defined physiology (i.e. human tissue equivalents), the complex 3-dimensional (3-D) tissue/organ physiology is captured in vitro, providing the opportunity to directly replace the use of animals in research/testing with more relevant systems. Therefore, it is imperative that testing strategies using organotypic models are developed that can address the limitations of current animal and cellular models and thus improve drug development, enabling faster delivery of drugs which are safer, more effective and have fewer side effects in humans.

Limitazioni dei modelli murini geneticamente modificati di malattie umane

[Bhogal N, Combes R. The relevance of genetically altered mouse models of human disease. Altern Lab Anim. 2006 Aug;34(4):429-54.]

Abstract: http://www.ncbi.nlm.nih.gov/pubmed/16945009

Full Text: http://www.frame.org.uk/dynamic_files/bhogal_comment.pdf

Nel testo:

“Limitations and problems

The generation of a single GA mouse strain can require the use of very large numbers of animals, because: a) the mutagenesis methods are inefficient; b) there is poor germline transmission of gene mutations; and c) genetic modification can adversely affect fecundity and survival. These problems are so acute that, in many studies, a large proportion of the animals are incidental to the work, since they are merely used for breeding or are offspring that fail to carry the desired mutation or lack a novel and detectable phenotype. Also, even if the desired GA mice are created, they are not always good models of human disease, for several fundamental reasons. These include the fact that humans are about 3000 times larger than mice; they possess a much greater number of cells, some of which have to communicate over long distances; and the lifespans of mice and humans are vastly different. Moreover, there is a lower likelihood that errors within the mouse genome will eventually result in chronic diseases such as cancer, due to the greatly reduced number of cell divisions that occur in the body of this species over its lifetime, as compared with humans (see Mouse modelsof cancer, DNA repair disorders and their use in toxicity testing).

We have stated earlier that the mouse and human genomes have similar gene clusters and mice possess gene homologues to many human genes. However, despite this, and despite the fact that the average similarity between mouse and human genes is 85%, with similarity ranging from 70–95% for specific genes, a single nucleotide difference can dramatically alter the function of the protein expressed. Such subtle changes can have important phenotypic consequences for a species, affecting physical characteristics, as well as biochemistry, physiology and pharmacology, leading via variations in temporal and spatial protein expression to species-specific metabolism, immune responses, sensory perception and endocrine functions.

Therefore, assumptions about the functional equivalence of homologous genes in mice and humans can be erroneous.

[…]

 “Are GA mice relevant and useful?

GA mouse models of human disease often lack relevance in the case of complex multigenic disorders.

Indeed, some studies in GA mice have been less informative than the corresponding investigations with less-complex organisms and cell culture systems.

This is particularly true for mouse models developed by using forward genetics, where an undefined number of mutations may have contributed to an overall phenotype which resembles a human disorder, but which may share few, if any, of the underlying biochemical or genetic causes of the respective human disorders. The relevance of many transgenic mouse models can be questioned on the basis that, even if a species gene homologue has the same function and expression patterns and levels in humans and mice, all the remaining components of the biochemical pathway must be equally represented in the surrogate animal, if relevant mouse models of human diseases are to be created within a laboratory setting.

Conclusioni:

Over the past decade, there has been a dramatic shift toward the use of GA mice in research and testing, which has, in turn, prompted concerns about the welfare of the animals used. Issues such as reducing the number of animals wasted during the production of GM mice, and the effective welfare assessment of GA mice, remain causes for concern. The main question is whether the widespread use of GA mice can be scientifically justified. Only by addressing this question objectively, and without bias, can we hope to identify the scope for replacing GA mice in research and testing. It should be recognised that the generation of GA mice has often confused, rather than improved, our understanding of the genetic basis of human diseases.The large numbers of only partly-relevant models available for many diseases have complicated the meaningful extrapolation of the information they provide to human medicine. There is an urgent need to re-evaluate GA mice as models of human disease, to take into account the availability of alternative models based on studies on lower organisms, normal and diseased human cells, and the increasingly availability of other sources of human information of direct relevance.

Cellule staminali pluripotenti indotte per studi su malattie genetiche

[Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-specific induced pluripotent stem cells. Cell. 2008 Sep 5;134(5):877-86. doi: 10.1016/j.cell.2008.07.041. Epub 2008 Aug 7.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633781/

Abstract:

Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research, but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance that include: adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21 and the carrier state of Lesch-Nyhan syndrome. Such patient-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.

Nel testo, critica al modello animale di sindrome di Down:

Murine models of human congenital and acquired diseases are invaluable but provide a limited representation of human pathophysiology. Murine models do not always faithfully mimic human diseases, especially for human contiguous gene syndromes such as trisomy 21 (Down syndrome or DS). A mouse model for the DS critical region on distal human chromosome 21 fails to recapitulate the human cranial abnormalities commonly associated with trisomy 21 (Olson et al., 2004). Orthologous segments to human chromosome 21 are present on mouse chromosomes 10 and 17 and distal human chromosome 21 corresponds to mouse chromosome 16 where trisomy 16 in the mouse is lethal (Nelson and Gibbs, 2004). Thus, a true murine equivalent of human trisomy 21 does not exist. […] 

For cases where murine and human physiology differ, disease-specific pluripotent cells capable of differentiation into the various tissues affected in each condition could undoubtedly provide new insights into disease pathophysiology by permitting analysis in a human system, under controlled conditions in vitro, using a large number of genetically-modifiable cells, and in a manner specific to the genetic lesions in each – whether known or unknown. Here, we report the derivation of human iPS cell lines from patients with a range of human genetic diseases.

Co-Colture 3D per ricapitolare la progressione dei tumori in vivo e per la medicina personalizzata

[Fang C, Man YG, Cuttitta F, Stetler-Stevenson W, Salomon D, Mazar A, Kulesza P, Rosen S, Avital I, Stojadinovic A, Jewett A, Jiang B, Mulshine J. Novel Phenotypic Fluorescent Three-Dimensional Co-Culture Platforms for Recapitulating Tumor in vivo Progression and for Personalized Therapy. J Cancer. 2013 Dec 1;4(9):755-763.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842444/

Figure 1

Abstract:

Because three-dimensional (3D) in vitro models are more accurate than 2D cell culture models and faster and cheaper than animal models, they have become a prospective trend in the biomedical and pharmaceutical fields, especially for personalized and targeted therapies. Because appropriate 3D models can be customized to mimic the in vivo microenvironment wherein various cell populations grow within an intricate but well organized extracellular matrix (ECM), they can accurately recapitulate physiological and pathophysiological progressions. The majority of cancers are carcinomas, which originate from epithelial cells, and dynamically interact with non-malignant cells including stromal cells (fibroblasts), vascular cells (endothelial cells and pericytes), immune cells (macrophages and mast cells), and the ECM. Employing a tumor monoclonal colony, tumor xenograft or patient cancer biopsy into an in vivo-like microenvironment, the native signaling pathways, cell-cell and cell-matrix interactions, and cell phenotypes are preserved and our fluorescent phenotypic 3D co-culture platforms can then accurately recapitulate the tumor in vivo scenario including tumor induced angiogenesis, tumor growth, and metastasis. In this paper, we describe a robust and standardized method to co-culture a tumor colony or biopsy with different cell populations, e.g., endothelial cells, immune cells, pericytes, etc. The procedures for recovering cells from the co-culture for molecular analyses, imaging, and analyzing are also described. We selected ECM solubilized extract derived from Engelbreth-Holm-Swam sarcoma cells. Because the 3D co-culture platforms can provide drug chemosensitivity data within 9 days that is equivalent to the results generated from mouse tumor xenograft models in 50 days, the 3D co-culture platforms are more accurate, efficient, and cost-effective and may replace animal models in the near future to predict drug efficacy, personalize therapies, prevent drug resistance, and improve the quality of life.